Toxoplasma gondii antigen Tg20

ABSTRACT

The present invention relates to isolated and pure Toxoplasma gondii antigenic fragments, recombinant polypeptides, nucleic acids encoding them, primers and probes derived from the same, as well as the use of these polypeptides, nucleic acids, primers and probes in methods and kits for the diagnosis and prevention of T. gondii infection in mammals (humans and animals).

This application is a 371 of PCT/EP97/00394 filed Jan. 27, 1997.

The present invention relates to isolated and pure Toxoplasma gondiiantigenic fragments, recombinant polypeptides, nucleic acids encodingthem, primers and probes derived from the same, as well as the use ofthese polypeptides, nucleic acids, primers and probes in methods andkits for the diagnosis and prevention of T. gondii infection in mammals(humans and animals).

Toxoplasma gondii is an ubiquitous intracellular protozoan parasitewhich infects mammals and birds. Although toxoplasmosis is in generalclinically asymptomatic in healthy individuals, it may cause severecomplications in pregnant women and immunocompromised patients. Ifprimary infection occurs during pregnancy, transplacental transmissioncan lead to abortion or neonatal malformations (for reviews seeRemington and Krahenbuhl, 1982; Hughes, 1985). In AIDS patients,Toxoplasma is recognized as a major opportunistic pathogen. In suchimmunodeficient individuals, rupture of cysts which persist in thetissues of the host after a primary infection and release ofproliferative forms of the parasite may cause severe disseminatedtoxoplasmosis and/or encephalitis. In approximately 30 percent ofToxoplasma-antibody-positive patients with AIDS, toxoplasmicencephalitis will develop due to reactivation of their latent infection.

The fetus and the newborn are very sensitive to toxoplasmosis. Infectionof the mother during pregnancy and transmission to the fetus can lead tomiscarriage, birth of abnormal children (especially with ocular andcerebral lesions), or birth of apparently normal children who willdevelop grave sequaelae months or years later (blindness, mentalretardation). Current estimates indicate that 0.1 to 0.9% of newbornsare afflicted with congenital toxoplasmosis.

Besides its negative impact on human health, the parasite is alsodetrimental in sheep and pig farming since abortions resulting from theinfection lead to relatively important economic losses (Beverly, 1976).

In the absence of efficient profylactic measures, there is a highnecessity for early, sensitive and specific diagnosis.

In response to infection, immunocompetent hosts mount an immuneresponse, which involves both humoral and cellular components (Remingtonand Krahenbuhl, 1982). The immune response confers protection againstsubsequent infection of the host: women showing serological evidence ofprevious infection before the onset of pregnancy are at no risk oftransmitting toxoplasmosis to their fetus. In-vitro and in-vivo studieshave indicated that cell-mediated immunity plays an essential role inprotection and have identified interferon-gamma (IFN-gamma) as the majormediator of resistance (Frenkel, 1967; Nathan et al., 1984; Pfefferkorn,1984; Sethi et al., 1985; Suzuki and Remington, 1988; Suzuki et al.,1988; Suzuki and Remington, 1990; Gazinelli et al., 1991).

The humoral component underlies the methods generally used intoxoplasmosis diagnosis. Like in other infectious diseases IgM classantibodies appear before IgG class antibodies. This difference is usedto know whether a pregnant woman has an acute or chronic phaseinfection, only the former being dangerous for fetal transmission.However, in a number of cases IgM class antibodies remain high for alonger than usual time period, making a differential diagnosisdifficult.

The reference tests in Toxoplasma diagnosis are the Toxoplasma lysistest (TLT) and the immunofluorescence test (IF). The detection ofantibodies against Toxoplasma is most often carried out by an EnzymeLinked Immunoassay (ELISA) or tests based on the same principle. Thespecificity and sensitivity of these tests is not always optimal anddepend on the quality of the antigen preparation used. Most often theantigen used is a fraction of a total cell lysate, lacking sufficientspecificity.

The use of a selection of well characterised Toxoplasma gondiirecombinant antigens could furnish better tools for a Toxoplasmadiagnostic assay.

Several antigens of Toxoplasma have already been cloned and expressed asrecombinant antigens. Several excreted/secreted antigens have been shownto be recognized by sera from patients: GRA1 (23 kDa) (Cesbron-Delauw etal, 1989; EP-A-0 346 430), GRA2 (28.5 or 28 kDa) (Mercier et al, 1993;S. F. Parmley et al, 1993; WO 93/25689), GRA6 (Lecordier et al, 1995),as well as some other cellular antigens, like SAG2 (P22) (Parmley et al,1992), SAG1 (P30) (Kim et al, 1994), ROP2 (54 kDa) (Van Gelder et al,1993) and a number of T. gondii antigenic fragments as described e.g. inEP-A-0 431 541 (Behringwerke).

A vaccine for controlling this infectious agent would be of great valueand the feasibility of its development is suggested by the fact thatprimary infection with Toxoplasma results in specific and long lastingimmunity against reinfection (Remington and Kranenbuhl, 1982). However,no effective and safe vaccine is currently available againsttoxoplasmosis in humans. A live temperature-sensitive mutant of thehighly virulent RH strain, can induce protective immunity in mice andhamsters. This mutant, names ts-4 does not persist in the host, as itcannot form bradyzoites and cysts (Waldeland and Frenkel, 1983; McLeodet al., 1988; Suzuki and Remington, 1990). Since 1988, a live vaccine isavailable for sheep. It consists of T. gondii tachyzoites of the S48incomplete strain grown on tissue culture (Toxovax, Ministry ofAgriculture and Fisheries, New Zealand). By passing through laboratorymice the strain lost the ability to develop bradyzoites (cysts). Thisvaccine protects naive sheep against an infect with T. gondii (Buxton,1991, 1993).

The aim of the present invention is to provide new polypeptides andpeptides useful in the diagnosis and/or profylaxis of Toxoplasma gondiiinfection in mammals.

It is more particularly an aim of the invention to provide polypeptidesand peptides useful in the serodiagnosis of T. gondii infection, andpossibly enabling discrimination between chronic and acute infection.

It is in addition an aim of the present invention to providepolypeptides and peptides useful in diagnostic assay for T. gondiiinfection based on the cellular immune response of the infected host.

It is moreover an aim of the present invention to provide forpolypeptides and peptides useful in a vaccine preparation against T.gondii infection.

It is a specific aim of the present invention to provide purified andisolated Tg20 antigenic fragments.

More specifically, it is an aim of the invention to provide recombinantTg20 antigenic fragments.

It is moreover an aim of the invention to provide the amino acidsequence of Tg20 antigenic fragments, and nucleic acid sequences codingfor the same.

It is moreover an aim of the invention to provide for monoclonal andpolyclonal antibodies specifically reacting with antigenic fragments ofthe Tg20 protein.

It is also an aim of the present invention to provide for primersspecifically amplifying Tg20 nucleic acid sequences, as well as probesspecifically hybridizing with Tg20 nucleic acid sequences.

It is another aim of the invention to provide for diagnostic methodsand/or kits for T. gondii infection, using the above-mentioned Tg20polypeptides, peptides, antibodies, primers and/or probes as one of theactive principles.

It is in particular an aim of the present invention to provide for aserodiagnostic method or kit for T. gondii infection, whereby the activeprinciple comprises the Tg20 polypeptides or peptides of the inventionin combination with other T. gondii antigens, more particularly, incombination with the Tg34 antigen (=Rop2 antigen), or fragments thereof,as described by Van Gelder et al. (1993).

It is finally an aim of the present invention to provide for a vaccinecomposition for providing protective immunity against T. gondiiinfection in mammals, more particularly in humans and/or domesticatedanimals.

All of the above-mentioned aims have been achieved by the followingembodiments of the invention.

As described in more detail in the examples section, the currentinvention describes the identification and sequencing of an antigenicfragment of T. gondii. Said antigenic fragment was selected from a λgt11expression library of T. gondii (clone Tg20), on the basis of itsreactivity with a number of sera from T. gondii infected patients, whichwere previously shown not to react with other T. gondii antigens, likethe Tg34 (Rop2) antigen, or fragments thereof (Van Gelder et al. 1993).

As described above, there is a current need to replace crude T. gondiiantigen preparations used in actual diagnostic assays, by a selection ofwell-defined isolated antigenic (poly)peptides. The Tg20 polypeptidesand peptides of the current invention are ideal candidates to beincluded in such “new generation” diagnostic assays, due to their highspecificity and sensitivity in detecting T. gondii infected individuals,as described further in the examples section. Moreover, the Tg20polypeptides have been selected such that they show a complementaryreactivity pattern with other T. gondii antigenic fragments, which havealready been described to be useful in diagnostics of toxoplasmosis,like the Tg34 antigen (Van Gelder et al. 1993). Tg20 and Tg34 antigenicpolypeptides are therefore particularly suitable to be used incombination with each other in a diagnostic assay.

The invention thus relates to a polypeptide or peptide containing

(1) an amino acid sequence extending from amino acid position x to aminoacid y in the sequence as shown in FIG. 1 b (SEQ ID NO 2), with

x=1 and y=196, and/or

x=1 and y=160, and/or

x=1 and y=145, and/or

x=95 and y=145, and/or

(2) any fragment of (1), with said fragment comprising a stretch of atleast 7 contiguous amino acids of the sequence as defined in (1), and/or

(3) any equivalents of (1) or (2) originating from the substitution ofone or several amino acids in the sequence of (1) or (2),

with said polypeptides or peptides containing a reactive epitopeimportant in the humoral and/or cell-mediated immune response of the T.gondii infected host organism.

Fragments of the above-mentioned polypeptides should contain at least 7contiguous amino acids, possibly also a stretch of 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 50 or morecontiguous amino acids selected from the amino acid sequence extendingfrom position 1 to position 196 as represented in FIG. 1 b (SEQ ID NO2).

Polypeptide fragments containing an epitope important in the serologicalresponse of an individual infected by T. gondii may be selected form ahydrophylicity plot of the antigen, according to principles known to theperson skilled in the art. The hydrophylicity plot of the Tg20 antigenof the invention (SEQ ID NO 2) is represented in FIG. 6. Theexperimental data in the examples section show that the mostimmunodominant epitopes of the Tg20 antigen are located in theaminoterminal part of the antigen, i.e. from amino acid position 1 toamino acid position 196, and more particularly in the region extendingfrom position 95 to position 145 of the sequence as represented in FIG.1. From the hydrophylicity plot of this Tg20 antigen (FIG. 6) it isclear however that additional epitopes may be located in the C-terminalpart of the protein, e.g. in the region extending from amino acidposition 197 to amino acid position 235, more particularly in the regionextending from position 205 to position 220.

The term “polypeptide” designates a linear series of amino acidsconnected one to the other by peptide bonds between the alpha-amino andcarboxy groups of adjacent amino acids. Polypeptides with a length oftwenty five amino acids or less can also be named “peptides”.Polypeptides can show a variety of lengths, either in their natural(uncharged) forms or in forms which are salts, and either free ofmodifications such as glycosylation, side chain oxidation, orphosphorylation or containing these modifications. It is well understoodin the art that amino acid sequences contain acidic and basic groups,and that the particular ionization state exhibited by the peptide isdependent on the pH of the surrounding medium when the protein is insolution, or that of the medium from which it was obtained if theprotein is in solid form. Also included in the definition are proteinsmodified by additional substituents attached to the amino acids sidechains, such as glycosyl units, lipids, or inorganic ions such asphosphates, as well as modifications relating to chemical conversions ofthe chains, such as oxidation of sulfhydryl groups.

It is to be understood that the polypeptides and peptides as describedin the current invention can also be extended at one or both of theirextremities by so called “linker” sequences, consisting of one orseveral amino acids (or other molecules, like e.g. biotine), andproviding additional physiochemical properties to the (poly)peptide,which may be particularly desirable for certain processes, like e.g.purification, coating and presentation of the (poly)peptide.

A special type of “linker” sequence is the addition of a biotin moleculeto the (poly)peptides of the invention. The biotin molecule may beincorporated in the polypeptide sequence either during or afterpolypeptide synthesis, and may be located N-terminal, or C-terminal orinternal of the polypeptide sequence. The biotinylation is especiallyadvantageous when the above-mentioned polypeptides are used inserodiagnostic assays: the presence of biotin in the (poly)peptidemolecule facilitates its adhesion and presentation on membranes orplates coated with a biotin binding molecule (like e.g. streptavidin).

Thus, “polypeptide” or its equivalent terms is intended to include theappropriate amino acid sequence referenced, subject to those of theforegoing modifications which do not destroy its functionality.

The polypeptides of the invention, and particularly the peptides, can beprepared by classical chemical synthesis.

The synthesis can be carried out in homogeneous solution or in solidphase.

For instance, the synthesis technique in homogeneous solution which canbe used is the one described by Houbenweyl in the book entitled“Methoden de Organischen Chemie” (Methods of Organic Chemistry) editedby E. Wunsh, vol. 15-I et II. THIEME, Stuttgart 1974.

The polypeptides of the invention can also be prepared in solid phaseaccording to the methods described by Atherton and Shepard in their bookentitled “Solid phase peptide synthesis” (IRL press, Oxford, 1989).

The polypeptides according to this invention can also be prepared bymeans of recombinant DNA techniques as described by Maniatis et al.,Molecular Cloning: A Laboratory Manual, New York, Cold Spring HarborLaboratory, 1982). The term “recombinant polypeptides” refers to apolypeptide produced by genetic engineering, through transcription andtranslation of a corresponding DNA sequence under the appropriateregulation elements within an efficient cellular host.

The polypeptides of the invention can also be prepared by isolation andpurification from their naturally occurring environment e.g. bypurification from T. gondii lysates or extracts. The techniques to beused for purification of the polypeptides of the invention are known inthe current art of protein purification.

The term “epitope” or “antigenic determinant” refers to a specificregion of an antigenic molecule that binds to an antibody (B-cellepitope) or to a T-cell receptor (T-cell epitope).

The term “humoral immune response” refers to the immune responsemediated by antibodies, while “cell mediated immune response” ismediated by T-lymphocytes. In the case of T. gondii infection, mostdiagnostic assays are based upon the measurement of the humoral immuneresponse (e.g. serodiagnosis), while protection towards the disease isbasically mediated by the cell-mediated immune response. Therefore, itcan be said that B-cell epitopes will be important components of adiagnostic assay, while T-cell epitopes will be indispensable in thecomposition of a vaccine.

As shown in the Examples section, the above-mentioned polypeptides andpeptides are selected such that they show a reactivity with sera from T.gondii infected individuals and/or stimulate T. gondii reactive T-cells.

The term “equivalents” (or “muteins”) as specified above, may be definedas polypeptides and peptides containing substitutions of one or severalamino acids, provided that said equivalents have retained theimmunogenic/antigenic properties of the original polypeptide. Anoverview of the amino acid substitutions which could form the basis forsuch equivalents is shown in Table 1. It should be evident that suchequivalents may have a slightly different molecular weight from theoriginal sequence as determined by SDS-PAGE.

Said “muteins” may be the result of strain to strain variations of theToxoplasma gondii Tg20 antigen, or may be the result of modificationsintroduced in the original polypeptide sequences, said modificationsbringing about a desirable side effect to the polypeptide molecule (e.g.better physiochemical properties, more efficient purification, moreefficient coating characteristics, more stable etc . . . ).

In a more specific embodiment, the invention relates to a polypeptide orpeptide containing

(1) the amino acid sequence extending from position 95 to position 145of the sequence in FIG. 1 b (SEQ ID NO 2), and/or

(2) any fragments of (1), with said fragments comprising a stretch of atleast 7 contiguous amino acids of the sequence in (1), and/or

(3) at least one of the amino acid sequences represented byGluProAspGluGlnGluGluValHisPheArgLysArgGlyValArgSerAspAlaGlu (SEQ ID NO3), or ArgLysArgGlyValArgSerAspAlaGluValThrAspAspAsnIleTyrGluGluHis (SEQID NO 4), orValThrAspAspAsnIleTyrGluGluHisThrAspArgLysValValProArgLysSer (SEQ ID NO5), or ThrAspArgLysValValProArgLysSerGluSlyLysArgSerPheLysAspLeuLeu (SEQID NO 6), or GluGlyLysArgSerPheLysAspLeuLeuLysLysLeuAlaLeuPro (SEQ ID NO7) and/or

(4) any equivalents of (1), (2) or (3) originating from the substitutionof one or several amino acids in the sequences of (1), (2) or (3),

with said polypeptides and peptides containing a reactive epitopeimportant in the humoral and/or cell-mediated immune response of the T.gondii infected host organism.

Preferably, the above-mentioned polypeptides and their fragments and/orequivalents are reactive with sera from T. gondii infected individuals.

Another embodiment of the invention encompasses a fusion proteinconsisting of one of the polypeptide sequences as defined above, linkedto a heterologous polypeptide sequence.

The term “heterologous polypeptide sequence” refers to any polypeptidesequence other than the Tg20 sequence itself, and may originate from T.gondii or from another organism.

A fusion protein may have advantageous effects over the non-fusionpolypeptides, said effects being e.g.

a more efficient expression level in a recombinant host (e.g. mTNFfusion protein as exemplified further) and/or,

a more easy purification system (e.g. polyhistidine tail as exemplifiedfurther) and/or,

a combination of several epitopes, originating from different T. gondiiantigens and/or,

a more efficient presentation of the epitopes.

Said fusion protein may be made by recombinant DNA technology, wherebydifferent coding regions are operably linked to each other and expressedin a suitable host cell. Fusion proteins may however also be preparedaccording to other methods, like in vitro coupling techniques, orchemical synthesis.

The invention also relates to nucleic acids containing

(1) a polynucleic acid sequence encoding any of the polypeptides asdefined above, and/or

(2) a polynucleic acid sequence extending from position x to position yof the polynucleic acid sequence as represented in FIG. 1 b (SEQ IDNO 1) with

x=1 and y=589, and/or

x=1 and y=480, and/or

x=1 and y=434, and/or

x=95 and y=434, and/or

(3) a polynucleic acid sequence which is degenerate as a result of thegenetic code to the polynucleic acid sequence of (1) or (2), and whichstill encodes any of the polypeptides as defined above, and/or

(4) a polynucleic acid sequence which hybridizes with any of thepolynucleic acid sequences as defined in (1) to (3), and/or

(5) a fragment of any of the polynucleic acid sequences defined in (1)to (4), said fragment containing a stretch of at least 10, andpreferably 11, 12, 13, 14, 15 or more contiguous nucleotides from any ofthe polynucleic acid sequences as defined in (1) to (3).

The term “nucleic acid” or “polynucleic acid” corresponds to eitherdouble-stranded or single-stranded cDNA or genomic DNA or RNA,containing at least 10, 20, 30, 40 or 50 contiguous nucleotides. Anucleic acid which is smaller than 100 nucleotides in length is oftenalso referred to as an oligonucleotide. Single stranded polynucleic acidsequences are always represented in the current invention from the 5′end to the 3′ end. It is to be understood however that the complementarysequences, and double stranded sequences are of course also encompassedby the formula used for presentation.

The term “hybridize to” refers to preferably stringent hybridizationconditions, allowing hybridization between sequences showing at least70%, 80%, 90%, 95% or more homology with each other.

The term “degenerate” refers to possible variations in the polynucleicacid sequence encoding the same protein, due to the possible occurrenceof different codons for the same amino acid.

According to another embodiment, the present invention relates to anoligonucleotide comprising in its sequence at least 10 contiguousnucleotides which form part of any of the polynucleic acid sequences asdefined above, for use as a specific hybridization probe for detectingthe polynucleic acids of the invention.

The term “probe” refers to single stranded sequence-specificoligonucleotides which have a sequence which is sufficientlycomplementary to hybridize to the target sequence to be detected.

Probes according to this aspect of the present invention may be chosenaccording to any of the techniques known in the art.

Preferably, these probes are about 10 to 50 nucleotides long, morepreferably from about 15 to 25 nucleotides.

It should be understood that the probe sequences of the invention may beslightly modified, e.g. by adding or deleting one or a few nucleotidesat the extremities (3′ or 5′) or by substituting some non-essentialnucleotides by others (including modified nucleotides and inosine), oncondition that these modifications do not alter the specificity of theprobes. These modifications may be necessary or desirable e.g. to obtaina higher sensitivity (e.g. because of strain variability of thesequences) or to obtain a sufficient specificity under modifiedhybridization conditions (buffer, temperature, salt concentration . . .). Also, changing the amount (concentration) of probe used may bebeneficial to obtain more specific hybridization results. It should benoted in this context, that probes of the same length, regardless oftheir GC content, will hybridize specifically at approximately the sametemperature in TMACl solutions (Jacobs et al., 1988).

The latter implies that variant probes contemplated within this aspectof the present invention can be defined as probes hybridizing with thesame specificity as the probe they are derived from under stringenthybridization and wash conditions, which may be different or the same asthe conditions used for the original probes.

The term “complement” refers to a nucleotide sequence which is exactlycomplementary to an indicated sequence and which is able to hybridize tothe indicated sequences.

According to another embodiment, the present invention relates to anoligonucleotide, comprising in its sequence at least 10 contiguousnucleotides which form part of any of the polynucleic acid sequences asdefined above, for use as a primer for specifically amplifying any ofthe polynucleic acid sequences of the invention.

The term “primer” refers to a single stranded DNA oligonucleotidesequence capable of acting as a point of initiation for synthesis of anextension product which is complementary to the nucleic acid strand tobe copies. The length and the sequence of the primer must be such thatthey allow to prime the synthesis of the extension products. Preferablythe primer is about 10-50 nucleotides long, more preferably 10-30nucleotides long. Specific length and sequence will depend on thecomplexity of the required DNA or RNA targets, as well as on theconditions of primer use such as temperature and ionic strength.

The fact that amplification primers do not have to match exactly withthe corresponding template sequence to warrant proper amplification isamply documented in the literature (Kwok et al., 1990).

The amplification method used can be either polymerase chain reaction(PCR; Saiki et al., 1988), ligase chain reaction (LCR; Landgren et al.,1988; Wu & Wallace, 1989; Barany, 1991), nucleic acid sequence-basedamplification (NASBA; Guatelli et al., 1990; Compton, 1991),transcription-based amplification system (TAS; Kwoh et al., 1989),strand displacement amplification (SDA; Duck, 1990; Walker et al., 1992)or amplification by means of Qβ replicase (Lizardi et al., 1988; Lomeliet al., 1989) or any other suitable method to amplify nucleic acidmolecules using primer extension. During amplification, the amplifiedproducts can be conveniently labelled either using labelled primers orby incorporating labelled nucleotides. Labels may be isotopic (³²P, ³⁵S,etc.) or non-isotopic (biotin, digoxigenin, etc.). The amplificationreaction is repeated between 20 and 80 times, advantageously between 30and 50 times.

The oligonucleotides used as primers or probes may also contain orconsist of nucleotide analoges such as phosphorothioates (Matsukura etal., 1987), alkylphosphoro-thiates (Miller et al., 1979) or peptidenucleic acids (Nielsen et al., 1991; Nielsen et al., 1993) or maycontain intercalating agents (Asseline et al., 1984).

As most other variations or modifications introduced into the originalDNA sequences of the invention, these variations will necessitateadaptions with respect to the conditions under which the oligonucleotideshould be used to obtain the required specificity and sensitivity.However, the eventual results of hybridization will be essentially thesame as those obtained with the unmodified oligonucleotides.

The introduction of these modifications may be advantageous in order topositively influence characteristics such as hybridization kinetics,reversibiltiy of the hybrid-formation, biological stability of theoligonucleotide molecules, etc.

Another embodiment of the invention provides for recombinant nucleicacids comprising any of the above-mentioned nucleic acids for cloningand/or expression purposes.

The term “recombinant nucleic acid” refers to a nucleic acid moleculewhich has been made in vitro by recombinant techniques, and which maycontain nucleic acids originating from different sources ligated to eachother.

In particular, the invention provides for a recombinant vector intowhich one of the above-mentioned nucleic acids has been inserted forcloning and/or expression purposes.

The term “vector” refers to an agent (virus or plasmid) used to transmitgenetic material to a cell or organism. A vector can be a “cloningvector” used to carry a fragment of DNA into a recipient cell for thepurpose of gene cloning, or an “expression vector” used to carry a DNAsequence into a suitable host cell and to direct the synthesis of theprotein encoded by the DNA sequence. In the current invention, the mostsuitable vectors are plasmids, i.e. small circular DNA molecules thatreplicate independently of the genome of the host cell. However, othervectors like cosmids, viruses and phages may also be used, depending onthe host cell used.

The present invention relates in particular to a recombinant vector intowhich the coding sequence for any of the polypeptides of the inventionis operably linked to a control sequence capable of providing theexpression of the coding sequence by the specific host.

The expression “operably linked to” refers to a juxtaposition where thecomponents are configured so as to perform their usual function. Thus,regulatory sequences operably linked to a coding sequence are capable ofdirecting the expression and eventually secretion of the coding gene.The term “control sequence” refers to those sequences involved in theexpression of a gene to protein i.e. its transcription and/ortranslation and/or regulation and/or maturation. Control sequences maythus comprise a promoter sequence, including possible promoterregulation sequences, a ribosome binding site, a sequence coding for asecretion signal, etc. Another example may be that the coding sequenceof the polypeptides of the invention is operably linked to another,heterologous coding sequence, being part of the vector sequence, andresulting in the expression of a fusion protein. Thus, in its broadestwording, the term “control sequence” may also comprise the codingsequence of a heterologous protein to which the polypeptides of theinvention are fused.

According to yet another embodiment, the present invention relates to ahost cell transformed by a recombinant vector comprising any of thepolynucleic acid sequences of the invention.

Preferably, said host cell is a bacterial host cell, most preferably E.coli, but it may also include eukaryotic cells like yeast, plant, insector animal cells.

The techniques for carrying out expression of the polypeptides of theinvention in E. coli are demonstrated further in the Examples section.The techniques for carrying out expression in any of the other hostcells are well known in the art of recombinant expression technology.

According to a subsequent embodiment, the invention provides for arecombinant polypeptide produced by

growing a culture of a transformed host cell as defined above, underconditions which allow the expression and possibly secretion of theencoded polypeptide, and

recovering the expressed polypeptide from the culture.

According to a possible embodiment, said recombinant polypeptide may bea fusion protein, consisting of the polypeptide of the invention fusedin frame to a heterologous (poly)peptide. As described above, a fusionprotein may bring about possible desired effect to the polypeptides ofthe invention.

Another embodiment of the invention provides for an antibody, monoclonalor polyclonal, reacting specifically with the polypeptides of theinvention.

Preferably, said antibody is different from the monoclonal antibody BATO214 which has been described earlier by Saavedra et al. (1990).

Antibodies according to this preferred embodiment of the inventioninclude specific polyclonal antisera prepared against the T. gondiipolypeptides of the invention, and showing no cross-reactivity to otherT. gondii proteins. It also includes monoclonal antibodies preparedagainst the T. gondii polypeptides of the invention.

The monoclonal antibodies of the invention can be produced by anyhybridoma formed according to classical methods known in the art, i.e.by fusion of splenic cells of an animal, particularly of a mouse or rat,infected with T. gondii or immunized against the polypeptides accordingto the invention defined above on the one hand, and of cells of amyeloma cell line on the other hand, and selected by the ability of thehybridoma to produce monoclonal antibodies recognizing the polypeptideswhich have been initially used for immunization of the animals.

The monoclonal antibodies according to a preferred embodiment of theinvention may be humanized versions of the mouse monoclonal antibodiesmade by means of recombinant DNA technology, departing from the mouseand/or human genomic DNA sequences coding for H and L chains or fromcDNA clones coding for H and L chains.

Also fragments derived from these monoclonal antibodies such as Fab,F(ab)′₂ and ssFv (“single chain variable fragment”), providing they haveretained the original binding properties, form part of the presentinvention. Such fragments are commonly generated by, for instance,enzymatic digestion of the antibodies with papain, pepsin, or otherproteases. It is well known to the person skilled in the art thatmonoclonal antibodies, or fragments thereof, can be modified for varioususes.

The antibodies involved in the invention can be labelled by anappropriate label of the enzymatic, fluorescent, or radioactive type.

The invention also relates to the use of the proteins of the invention,muteins thereof, or fragments thereof, for the selection of recombinantantibodies by the process of repertoire cloning (Perrson et al., 1991).

According to a preferred embodiment of the present invention, anantibody, or an antigen-binding fragment F(ab′)₂, Fab, single chain Fvand all types of recombinant antibodies, as defined above are furthercharacterized in that they can inhibit the infection by Toxoplasmagondii of the specific cell type which it infects in vivo.

According to another embodiment, the present invention relates to amonoclonal antibody as defined above, obtainable by a process comprisingat least the following steps:

fusing the splenocytes from mice infected with Toxoplasma gondiitogether with myeloma cells, and

selecting the anti-T. gondii hybridomas by means of ELISA and subsequentlimiting dilution,

selecting the hybridomas producing a monoclonal antibody, specificallydirected against any of the T. gondii polypeptides of the invention bymeans of ELISA, and,

recovering the monoclonal antibodies from ascites fluid or from aculture of the selected hybridomas.

The present invention also relates to a hybridoma producing any of themonoclonal antibodies as defined above.

The present invention further relates to an anti-idiotype antibodyraised against any of the antibodies as defined above.

The term “anti-idiotype antibodies” refers to monoclonal antibodiesraised against the antigenic determinants of the variable region ofmonoclonal antibodies themselves raised against the Toxoplasma Tg20polypeptides. These antigenic determinants of immunoglobulins are knownas idiotypes (sets of idiotopes) and can therefore be considered to bethe “fingerprint” of an antibody (for review see de Pr{acute over(e)}val, 1978; Fleishmann and Davie, 1984). The methods for productionof monoclonal anti-idiotypic antibodies have been described by Gheuensand McFarlin (1982). Monoclonal anti-idiotypic antibodies have theproperty of forming an immunological complex with the idiotype of themonoclonal antibody against which they were raised. In this respect themonoclonal antibody is often referred to as Ab1, and the anti-idiotypicantibody is referred to as Ab2. These anti-idiotype Ab2s may be used assubstitutes for the polypeptides of the invention or as competitors forbinding of the polypeptides of the invention to their target.

The present invention further relates to antisense peptides derived fromthe Toxoplasma polypeptides as defined above.

More particularly, the term “antisense peptide” is reviewed by Blalock(1990) and by Roubos (1990). In this respect, the molecular recognitiontheory (Blalock, 1990) states that not only the complementary nucleicacid sequences interact but that, in addition, interacting sites inproteins are composed of complementary amino acid sequences (senseligand with receptor or sense ligand with antisense peptides). Thus, twopeptides derived from complementary nucleic acid sequences in the samereading frame will show a total interchange of their hydrophobic andhydrophilic amino acids when the amino terminus of one is aligned withthe carboxy terminus of the other. This inverted hydropathic patternmight allow two such peptides to assume complementary conformationsresponsible for specific interaction.

The antisense peptides can be prepared as described in Ghiso et al.(1990). By means of this technology it is possible to logicallyconstruct a peptide having a physiologically relevant interaction with aknown peptide by simple nucleotide sequence analysis forcomplementarity, and synthesize the peptide complementary to the bindingsite.

The present invention still further relates to a method for in vitrodiagnosis of T. gondii infection comprising at least the step ofcontacting a sample possibly containing anti-T. gondii antibodies, T.gondii antigens and/or T. gondii nucleic, with:

a polypeptide or peptide as defined above, under conditions allowing theformation of an immunological complex, or,

a probe as defined above, under conditions allowing the formation of ahybridization complex, with said nucleic acids of said sample beingpossibly amplified prior to hybridization, using a primer as definedabove, or,

an antibody specifically directed against a polypeptide as definedabove, under conditions allowing the formation of an immunologicalcomplex, or,

an anti-idiotype antibody as defined above, under conditions allowingthe formation of an antibody-anti-idiotypic complex, or,

an antisense peptide as defined above, under conditions allowing theformation of an antigen-antisense peptide complex,

and subsequently detecting the complexes formed.

The term “sample” may refer to any biological sample (tissue or fluid)containing T. gondii nucleic acids, antibodies or polypeptides.

In a preferential embodiment, the invention relates to a method fordetection of anti-T. gondii antibodies and the preferred sample in thatcase is serum or plasma.

In a more specific embodiment, the invention relates to a method fordetecting antibodies to T. gondii present in a biological sample,comprising:

contacting the biological sample to be analysed with any of thepolypeptide as described above, under conditions allowing the formationof an immunological complex, and

detecting the immunological complex formed between said antibodies andsaid polypeptide.

Conditions allowing the formation of an immunological complex are knownto the person skilled in the art.

In a special embodiment, the polypeptides being used in theabove-described method for detection of anti-T. gondii antibodies, canbe replaced by anti-idiotype antibodies as described above, acting astheir equivalents.

Conditions allowing the formation of an antibody-anti-idiotypic complexare known in the art.

The invention further relates to a method for detecting the presence ofT. gondii antigens in a biological sample, comprising:

contacting the biological sample to be analysed with an antibodyaccording to the invention, under conditions allowing the formation ofan immunological complex, and

detecting the immunological complex formed between said antigens andsaid antibody.

In a special embodiment, the antibodies being used in theabove-described method for detection of T. gondii antigens, may bereplaced by anti-sense peptides as described above, acting as theirequivalents.

Conditions allowing the formation of an antigen-antisense peptidecomplex are known in the art.

Design of immunoassays is subject to a great deal of variation, and manyformats are known in the art. Protocols may, for example, use solidsupports, or immunoprecipitation. Most assays involve the use of labeledantibody or polypeptide; the labels may be, for example, enzymatic,fluorescent, chemiluminescent, radioactive, or dye molecules. Assayswhich amplify the signals from the immune complex are also known;examples of which are assays which utilize biotin and avidin orstreptavidin, and enzyme-labeled and mediated immunoassays, such asELISA assays.

An advantageous embodiment provides for a method for detection ofanti-T. gondii antibodies in a sample, whereby the (poly)peptides of theinvention are immobilized on a solid support, eventually on a membranestrip. Different (poly)peptides of the invention may be immobilizedtogether or next to each other on distinct locations (e.g. in the formof parallel lines). The polypeptides of the invention may also becombined in the same assay with other antigens from Toxoplasma gondii orfrom other organisms.

The invention thus also relates to a solid support onto which the(poly)peptides of the invention, possibly in combination with other(poly)peptides, have been immobilized.

Another embodiment of the invention provides for a method for detectingthe presence of T. gondii polynucleic acids present in a biologicalsample, comprising:

possibly extracting the polynucleic acids contained in the sample,

amplifying the T. gondii polynucleic acids with at least one primer asdescribed above,

detecting the amplified nucleic acids, possibly after hybridization witha probe as described above.

Conditions allowing hybridization are known in the art and e.g.exemplified in Maniatis et al. (1982). However, according to thehybridization solution (SSC, SSPE, etc.), the probes used should behybridized at their appropriate temperature in order to attainsufficient specificity (in some cases differences at the level of onenucleotide mutation are to be discriminated).

Amplification of nucleic acids present in a sample prior to detection invitro may be accomplished by first extracting the nucleic acids presentin the sample according to any of the techniques known in the art. Incase of extraction of RNA, generation of cDNA is necessary; otherwisecDNA or genomic DNA is extracted.

The amplification methods are detailed above.

Suitable assay methods for purposes of the present invention to detecthybrids formed between oligonucleotide probes according to the inventionand the nucleic acid sequences in a sample may comprise any of the assayformats kown in the art. For example, the detection can be accomplishedusing a dot blot format, the unlabelled amplified sample being bound toa membrane, the membrane being incubated with at least one labelledprobe under suitable hybridization and wash conditions, and the presenceof bound probe being monitored. Probes can be labelled withradioisotopes or with labels allowing chromogenic or chemilumeniscentdetection such as horse-radish peroxidase coupled probes.

An alternative is a “reverse” dot-blot format, in which the amplifiedsequence contains a label. In this format, the unlabelledoligonucleotide probes are bound to a solid support and exposed to thelabelled sample under appropriate stringent hybridization and subsequentwashing conditions. It is to be understood that also any other assaymethod which relies on the formation of a hybrid between the nucleicacids of the sample and the oligonucleotide probes according to thepresent invention may be used.

According to an advantageous embodiment, the process of detecting T.gondii nucleic acid sequences contained in a biological sample comprisesthe steps of contacting amplified fragments of the polynucleic acids ofthe invention, with a solid support onto which probes as defined above,have been previously immobilized.

In a very specific embodiment, the probes have been immobilized on amembrane strip in the form of parallel lines. This type of reversehybridization method is specified further as a Line Probe Assay (LiPA).

The invention thus also relates to a solid support onto which thepolynucleotides of the invention have been immobilized.

According to another embodiment, the present invention relates to amethod using at least one of the polypeptides or peptides of theinvention, possibly in combination with other T. gondii polypeptides,for measuring a cellular immune response in an individual which has beenin contact with the T. gondii pathogen, said cellular immune responsebeing measured either in vivo, such as a delayed type hypersensitivityreaction upon subcutaneous injection of the polypeptides of theinvention, or in vitro, such as stimulation of periferal bloodlymphocytes of secretion of interferon-gamma, upon addition of thepolypeptides of the invention to a sample of periferal blood lymphocytesunder conditions allowing recognition of the polypeptides by the cellsresponsive for the immune response, conditions which are known to theperson skilled in the art.

The present invention also relates to a kit for detecting anti-T. gondiiantibodies, or T. gondii nucleic acids or T. gondii antigens accordingto any of the methods as defined here above, comprising at least one ofthe T. gondii polypeptides or peptides as defined above, or any of theT. gondii nucleic acids as defined above, more specifically any of theprobes or primers as defined above, or any of the antibodies as definedabove, or any of the anti-idiotypic antibodies as defined above, or anyof the antisense-peptides as defined above.

According to this embodiment, the detection of antibodies against the T.gondii polypeptides of the invention is preferred over the detection ofnucleic acids of polypeptides.

The present invention relates more particularly to a kit for determiningthe presence of anti-T. gondii polypeptide antibodies as defined above(=a serodiagnostic kit/assay) in a biological sample, comprising:

at least one polypeptide or peptide as defined above, possibly incombination with other polypeptides or peptides from T. gondii, withsaid polypeptides being preferentially immobilized on a solid substrate,

a buffer, or components necessary for producing the buffer, enabling abinding reaction between the polypeptides of the invention and theantibodies possibly present in the biological sample,

means for detecting the immune complexes formed in the preceding bindingreaction,

possibly also including an automated scanning and interpretation devicefor inferring the presence of T. gondii antibodies in the sample.

The kit or method according to this aspect of the present invention maycomprise in addition to peptide or polypeptide antigens according to theinvention, also other T. gondii antigenic proteins or peptides known inthe art (such as outer membrane protein (OMP) proteins or peptides), orother bacterial antigenic proteins or peptides in general.

The combination of different antigens in one single detection method orkit as described above, may have certain advantages, such as forexample:

achieving a higher test sensitivity: e.g. by combining several antigenicdeterminants from T. gondii, the number of correctly identified positivesera may be greater, and/or

enabling differentiation between chronic and acute T. gondii infection.

In a preferential embodiment, the invention relates to a serodiagnosticmethod and/or kit as described above, whereby the polypeptides andpeptides of the invention are combined with the Tg34 antigen of T.gondii, or fragments thereof, as described by van Gelder et al. 1993.

Another particular case of combination, also in included in the currentinvention, is the combination of the polypeptides of the currentinvention with antigenic fragments located in the C-terminal part of theTg20-protein, i.e. sequences located in the region from amino acidposition 197 to amino acid position 235 of the sequence represented inFIG. 1 b (SEQ ID NO 2). As described further in the examples section,the most immunodominant epitopes of the Tg20 protein reside in theregion extending from amino acid position 1 to amino acid position 197,more particularly in the region extending from position 95 to position145 as represented in FIG. 1 b. However, it may be necessary, in certaininstances, to combined the polypeptides of the invention with(poly)peptide fragments originating from the less antigenic C-terminalpart of Tg20.

Said combination of antigens in one assay may be accomplished indifferent ways, depending on the application or test format. Forexample, antigenic fragments of several polypeptides (e.g. Tg20 of theinvention and Tg34) may be immobilized together on a solid surface(microtiter plate or membrane . . . ), in the same location or indistinct locations. A preferred example of the latter test format is aLine Immuno Assay (LIA, INNOGENETICS) where different antigenicfragments are immobilized as parallel lines on a membrane strip, thusallowing to monitor the differential reactivities with the differentantigenic fragments.

Alternatively, the combination of the polypeptides of the invention withother T. gondii antigenic determinants, may be made on the moleculelevel, i.e. by the creation of a “superantigen” for the detection of T.gondii infection, combining different antigenic epitopes in onemolecule. Said “superantigen” may be produced by recombinant DNAtechniques, known in the art, e.g. by linking operably together thedifferent DNA sequences encoding the different antigenic sites andexpressing them in a suitable host.

It is to be understood that the above-described combination is notexclusively to be used in serodiagnostic assays, but may also prove tobe useful in other types of diagnostic assays, and/or in vaccinecompositions.

In a very specific embodiment the invention relates to a kit for thedetection of anti-T. gondii antibodies in a biological sample asdescribed above, whereby the polypeptides of the invention are replacedby the anti-idiotype antibodies as described above.

The invention further relates to a diagnostic kit for the detection ofantigens of T. gondii present in a biological sample, said kitcomprising an antibody as described above, with said antibody beingpreferably bound to a solid support.

In a very specific embodiment, the invention relates to a diagnostic kitfor the detection of antigens of T. gondii present in a biologicalsample, whereby the antibody as described above is replaced by anantisense peptide.

The invention further also relates to a diagnostic kit for the detectionof T. gondii polynucleic acids present in a sample, said kit comprisinga probe as described above and/or a primer as described above.

According to a preferred embodiment, the present invention relates to akit or method for diagnosis of T. gondii infection as defined above,further characterized in that said polypeptides, peptides, polynucleicacids, antibodies, anti-idiotypic antibodies or anti-sense peptides areparticularly useful for differentiating in vitro T. gondii chronicallyinfected individuals from acutely infected individuals.

According to another embodiment, the present invention relates to avaccine composition which provides protective immunity against T. gondiiinfection comprising as an active principle one or more of the T. gondiipolypeptides according to the invention, or one or more of thepolynucleic acid sequences or recombinant vectors according to theinvention, said active principle being combined with a pharmaceuticallyacceptable carrier.

According to a special embodiment, the vaccine composition as describedabove may comprise as an active principle one of the anti-idiotypeantibodies as described above.

Besides the T. gondii proteins according to the invention, said vaccinecomposition may also comprise any other Toxoplasma immunogeniccomponents (such as Rop2 (Tg34), Gra1, Gra2, Sag1, Sag2 antigens e.o.)or any other bacterial or other immunogenic components in general.

In a specific embodiment, polynucleic acid sequences coding for any ofthe polypeptides as defined above, are used as a vaccine, either asnaked DNA or as part of recombinant vectors. In this case, it is the aimthat said nucleic acids which are administered to the individual to beimmunized, are expressed into immunogenic protein/peptide in situ andthus confer protection to the vaccinated host (e.g. Ulmer et al., 1993).

The active ingredients of such a vaccine composition may be administeredorally, subcutaneously, conjunctivally, intramuscularly, intranasally,or via any other route known in the art including for instance via thebinding to carriers, via incorporation into liposomes, by addingadjuvants known in the art, etc.

The invention also relates to any of the above-mentioned substances(polypeptides, antibodies, polynucleic acids, anti-idiotype antibodies,antisense peptides) for use as a medicament, more particularly for anyof the medical (diagnostic or polphylactic) applications as mentionedabove.

Furthermore, the invention relates to the use of any of theabove-mentioned substances (polypeptides, antibodies, polynucleic acids,anti-idiotype antibodies, antisense peptides) for the manufacture of amedicament, more particularly for the preparation af a vaccine or forthe preparation of a diagnostic composition.

FIGURE LEGENDS

FIG. 1

1A Restriction map of the 1260 bp EcoRI fragment of clone Tg20 andposition of the different deletion clones analyzed for immunereactivity.

1B-1D Complete nucleic acid (SEQ ID NO 1) and amino acid (SEQ ID NO 2)sequence of the 1260 bp EcoRI fragment of clone Tg20. Relevantrestriction sites are indicated.

Arrows indicate possible signal peptide cleavage sites.

FIG. 2 Map of vector pm TNF.MPH

FIG. 3 Map of vector pIGFH111

FIG. 4 Amino acid sequence of mTNF.H6.Tg20 fusion protein. Normalcharacters represent the amino acid sequence originating from thevector. Bold characters correspond to the Tg20 antigen sequence.

FIG. 5 Reactivity in Western Blot with Mab BATO 214 of the recombinantTg20 antigen and is naturally occurring counterpart.

Lane 1: T. gondii lysate

Lane 2: MW markers (Biolabs) with increasing sizes of 6.5, 16.5, 25.0,32.5, 47.5, 62.0, 83.0, 175.0 kDa.

Lane 3: purified recombinant Tg20 fusion protein.

FIG. 6 Hydrophylicity plot of the Tg20 antigen (SEQ ID NO 2).

TABLE LEGENDS

Table 1: Amino acid substitutions which may form the basis for themuteins (equivalents) according to the present invention.

Table 2: Reactivity (OD450 values×1000) of different (human) sera in theclassical tests used for Toxoplasmosis diagnosis (IF (IgG and IgM) andBiom{acute over (e)}rieux ELISA test), and in ELISA using Tg20 antigenof the invention, or a fragment thereof (Tg20BN), in ELISA using theTg34AR antigen, or combinations of the foregoing antigens.

Sera positive in classical testing are numbered from 1 to 95.

Sera negative in classical testing are numbered from 206 to 250.

Table 3: Comparison of reactivity of some of the sera from table 2 withthe full size Tg20 antigen and the Tg20 fragment.

TABLE 1 Amino acids Synonymous groups Ser (S) Ser, Thr, Gly, Asn Arg (R)Arg, His, Lys, Glu, Gln Leu (L) Leu, Ile, Met, Phe, Val, Tyr Pro (P)Pro, Ala, Thr, Gly Thr (T) Thr, Pro, Ser, Ala, Gly, His, Gln Ala (A)Ala, Pro, Gly, Thr Val (V) Val, Met, Ile, Tyr, Phe, Leu, Val Gly (G)Gly, Ala, Thr, Pro, Ser Ile (I) Ile, Met, Leu, Phe, Val, Tyr Phe (F)Phe, Met, Tyr, Ile, Leu, Trp, Val Tyr (Y) Tyr, Phe, Trp, Met, Ile, Val,Leu Cys (C) Cys, Ser, Thr, Met His (H) His, Gln, Arg, Lys, Glu, Thr Gln(Q) Gln, Glu, His, Lys, Asn, Thr, Arg Asn (N) Asn, Asp, Ser, Gln Lys (K)Lys, Arg, Glu, Gln, His Asp (D) Asp, Asn, Glu, Gln Glu (E) Glu, Gln,Asp, Lys, Asn, His, Arg Met (M) Met, Ile, Leu, Phe, Val

TABLE 2 Serum IF IgG IF IgM BioMer Tg34AR Tg34AR Nr titre titre IgGTg34AR Tg20 Tg20 Tg20BN 1 1/256  1/50 1219 378 524 849 795 2 1/64  <1/50616 702 518 1105 1101 3 1/64  <1/50 1085 197 147 286 251 4 1/64  <1/50337 23 33 107 73 5 1/256  1/50 716 430 119 586 674 6 1/64  <1/50 598 87499 664 845 7 1/64  <1/50 988 487 103 653 871 8 1/64  <1/50 660 183 54433 381 9  1/1024 <1/50 809 1473 429 1608 1821 10 1/256 <1/50 952 507257 685 712 11 1/256 <1/50 1014 201 117 252 301 12 1/64  <1/50 1014 678344 1048 1074 13 1/256 <1/50 1102 313 375 474 603 14 1/64  876 652 10101443 1304 15 1/256 1093 260 983 1049 564 16 1/64  956 213 183 435 439 171/64  <1/50 262 85 136 195 192 18  1/1024 <1/50 1153 1058 1413 1904 185619 1/64  <1/50 510 142 8 533 401 20 1/256 <1/50 1381 678 669 943 1124 211/64  <1/50 768 108 249 493 340 22  1/1024  1/50 1361 692 748 1401 119623 1/256 <1/50 1434 861 374 1079 1152 24  1/1024 <1/50 1262 506 283 812916 25  1/1024  1/100 1295 1848 723 2030 2096 26 1/256 <1/50 1137 303357 849 593 27  1/1024 1495 526 649 1344 1087 28  1/1024 <1/50 1593 8822346 2105 1925 29 1/256 1415 274 1430 1377 1199 30  1/1024 <1/50 1391593 1248 1305 1089 31 1/256 1221 283 1589 1527 1328 32 1/64  <1/50 17334 127 213 137 33 1/256 <1/50 530 42 58 137 141 34 1/256 <1/50 498 29342 406 303 35 1/256 <1/50 1273 444 336 921 578 36 1/256 <1/50 873 298200 384 435 37 1/256 <1/50 1074 748 430 1193 1205 38 1/256 1351 475 11541397 1106 39 1/64  <1/50 157 31 48 67 62 40 1/64  <1/50 591 1360 1410869 426 41 1/64  <1/50 663 175 176 325 378 42 1/256 <1/50 1388 595 16401711 1624 43 1/64  <1/50 697 139 127 369 279 44 1/256  1/200 1323 2931226 1442 1199 45 1/64  <1/50 237 42 32 49 37 46 1/256 <1/50 789 226 39426 472 47 1/64  387 115 442 504 547 48  1/1024 <1/50 1224 1244 11151765 1940 49  1/1024 <1/50 648 268 366 563 448 50 1/256 <1/50 646 256492 697 611 51  1/1024 <1/50 1351 348 845 1102 636 52 1/256 <1/50 589489 280 480 492 53 1/256 <1/50 355 25 75 118 98 54  1/1024 <1/50 11861727 1199 1874 1891 55 1/256 <1/50 1180 736 1840 1836 1723 56  1/1024 1/50 1367 1542 2515 2114 1929 57 1/256  1/50 569 164 1221 972 400 58 1/1024 832 356 421 585 486 59 1/256 <1/50 959 270 909 778 431 60 1/1024  1/100 1498 1900 2669 2216 2307 61 1/256 <1/50 1041 239 160 514396 62 1/256 <1/50 324 64 34 68 48 63 1/64  <1/50 170 96 64 68 53 641/256 902 192 901 767 334 65 1/256 <1/50 1428 776 1189 1609 1455 66 1/1024 <1/50 1495 1216 1559 1764 1856 67 1/256 <1/50 1042 1115 364 11541222 68 1/64  <1/50 489 91 50 139 127 69 1/64  <1/50 1134 1015 1493 18151817 70 1/256 <1/50 1247 322 721 1280 1004 71  1/1024 <1/50 1352 1170529 1300 1382 72 1/256 <1/50 666 423 393 808 753 73 1/64  <1/50 673 17636 110 141 74 1/256 613 457 712 962 627 75  1/1024 1001 1290 302 16871720 76 1/64  <1/50 258 8 12 33 19 77 1/256  1/100 1224 330 972 1197 90978 1/256 <1/50 1167 665 271 974 891 79 1/64  <1/50 462 37 57 121 109 801/256 <1/50 1419 677 1067 1338 1165 81 1/256  1/50 1151 284 377 642 56982 1/256 <1/50 734 224 24 22 10 83 1/64  <1/50 617 108 204 344 145 841/64  <1/50 134 61 66 96 62 85 1/256 <1/50 1203 1546 21 821 1377 861/64  <1/50 239 95 243 383 356 87 1/64  <1/50 419 56 310 410 295 881/256 <1/50 1288 783 370 992 888 89 1/256 <1/50 1188 428 316 599 573 901/256 <1/50 350 82 102 198 123 91  1/1024 <1/50 1451 279 1051 1186 91092 1/256 <1/50 1151 536 424 819 854 93 1/256 <1/50 1198 470 215 861 64494 1/256 <1/50 911 532 523 729 593 95 1/256 <1/50 770 432 211 420 406206 <1/64  <1/50 19 1 10 10 7 207 <1/64  <1/50 30 3 10 17 7 208 <1/64 <1/50 37 53 18 18 10 209 <1/64  <1/50 52 0 59 80 36 210 <1/64  <1/50 310 13 16 8 211 <1/64  <1/50 12 50 19 20 11 212 <1/64  <1/50 17 11 14 1510 213 <1/64  <1/50 14 5 12 13 10 214 <1/64  <1/50 24 18 19 15 10 215<1/64  <1/50 14 0 12 13 8 216 <1/64  <1/50 25 34 37 26 18 217 <1/64 <1/50 40 14 20 17 12 218 <1/64  <1/50 37 3 12 12 10 219 <1/64  <1/50 1225 142 86 35 220 <1/64  <1/50 15 4 19 19 12 222 <1/64  <1/50 22 122 1111 5 223 <1/64  <1/50 43 12 10 10 7 224 <1/64  <1/50 39 5 25 25 14 225<1/64  <1/50 61 15 59 24 8 226 <1/64  <1/50 26 12 37 24 7 227 <1/64 <1/50 30 4 15 12 8 228 <1/64  <1/50 35 257 16 66 100 229 <1/64  <1/50 286 36 39 9 230 <1/64  <1/50 31 5 11 10 6 231 <1/64  <1/50 27 3 9 12 7 232<1/64  <1/50 29 5 36 35 21 234 <1/64  <1/50 38 4 24 17 10 235 <1/64 <1/50 30 9 21 38 11 236 <1/64  <1/50 33 16 16 20 15 237 <1/64  <1/50 4527 59 38 9 238 <1/64  <1/50 29 2 23 26 5 239 <1/64  <1/50 24 0 32 51 116240 <1/64  <1/50 38 5 13 11 8 241 <1/64  <1/50 30 5 17 10 11 242 <1/64 <1/50 29 15 15 30 7 243 <1/64  <1/50 23 1 12 16 8 244 <1/64  <1/50 34 2136 125 11 245 <1/64  <1/50 25 1 12 16 7 246 <1/64  <1/50 27 35 19 13 6247 <1/64  <1/50 27 0 12 12 8 248 <1/64  <1/50 25 1 13 13 8 249 <1/64 <1/50 26 2 45 15 9 250 <1/64  <1/50 29 5 30 21 10

TABLE 3 Serum IF IgG IF IgM BioMer Nr titre titre IgG Tg20 Tg20BN 11/256  1/50 1219 714 703 2 1/64  <1/50 616 746 607 3 1/64  <1/50 1085203 130 4 1/64  <1/50 337 71 42 5 1/256  1/50 716 225 124 6 1/64  <1/50598 175 91 7 1/64  <1/50 988 209 137 8 1/64  <1/50 660 95 59 9  1/1024<1/50 809 570 297 10 1/256 <1/50 952 380 196 11 1/256 <1/50 1014 146 17312 1/64  <1/50 1014 529 393 13 1/256 <1/50 1102 374 253 14 1/64  8761416 1108 15 1/256 1093 1194 350 16 1/64  956 286 160 17 1/64  <1/50 262235 159 18  1/1024 <1/50 1153 1776 1050 19 1/64  <1/50 510 510 278 201/256 <1/50 1381 268 185 21 1/64  <1/50 768 335 141 22  1/1024  1/501361 985 639 23 1/256 <1/50 1434 532 309 24  1/1024 <1/50 1262 435 27825  1/1024  1/100 1295 974 594 26 1/256 <1/50 1137 369 328 27  1/10241495 736 553 28  1/1024 <1/50 1593 2449 2270 29 1/256 1415 1612 1320 30 1/1024 <1/50 1391 1242 1013 31 1/256 1221 1476 1612 32 1/64  <1/50 173168 166 33 1/256 <1/50 530 148 86 34 1/256 <1/50 498 324 379 35 1/256<1/50 1273 578 75 36 1/256 <1/50 873 242 244 37 1/256 <1/50 1074 490 44738 1/256 1361 1268 900 30 1/64  <1/50 157 91 45 40 1/64  <1/50 591 112884 41 1/64  <1/50 663 167 161 42 1/256 <1/50 1388 1701 1538 43 1/64 <1/50 697 195 80 44 1/256  1/200 1323 1620 1239 45 1/64  <1/50 237 54 7046 1/256 <1/50 789 68 49 47 1/64  387 623 534 48  1/1024 <1/50 1224 1448844 49  1/1024 <1/50 648 390 290 50 1/256 <1/50 646 712 470 206 <1/64 <1/50 19 18 13 207 <1/64  <1/50 30 17 10 208 <1/64  <1/50 37 31 22 209<1/64  <1/50 52 210 <1/64  <1/50 31 24 14 211 <1/64  <1/50 12 31 15 212<1/64  <1/50 17 36 27 213 <1/64  <1/50 14 21 15 214 <1/64  <1/50 24 5713 215 <1/64  <1/50 14 19 10 216 <1/64  <1/50 25 20 15 217 <1/64  <1/5040 43 20 218 <1/64  <1/50 37 15 10 219 <1/64  <1/50 12 163 143 220<1/64  <1/50 15 39 28 222 <1/64  <1/50 22 17 10 223 <1/64  <1/50 43 2914 224 <1/64  <1/50 39 71 80 225 <1/64  <1/50 61 73 82 226 <1/64  <1/5026 37 22 227 <1/64  <1/50 30 22 10 229 <1/64  <1/50 28 32 13 230 <1/64 <1/50 31 16 9 231 <1/64  <1/50 27 12 10 232 <1/64  <1/50 29 53 96 234<1/64  <1/50 38 54 71 235 <1/64  <1/50 30 46 64 236 <1/64  <1/50 33 2915 237 <1/64  <1/50 45 60 59 238 <1/64  <1/50 29 37 18 239 <1/64  <1/5024 67 95 240 <1/64  <1/50 38 23 12 241 <1/64  <1/50 30 32 36 242 <1/64 <1/50 29 24 13 243 <1/64  <1/50 23 21 22 244 <1/64  <1/50 34 117 22 245<1/64  <1/50 25 18 15

EXAMPLES Example 1: Materials and Methods

Reagents.

All reagents were of analytical grade and obtained from Merck(Darmstadt, Germany), Sigma (St. Louis, Mo.) or Bio-Rad Laboratories(Richmond, Calif.). Restriction enzymes and DNA modifying enzymes werepurchased from Boehringer Mannheim (Brussels, Belgium) and were usedaccording to the manufacturer's instructions. Protein concentrationswere determined by the bicinchoninic acid method (Pierce, Rockford,Ill.)

Monoclonal antibodies.

Monoclonal antibody BATO 214 was prepared as described in Saavedra etal., (1990). Anti-TNF monoclonal was produced from hybridoma culturesupernatant.

Human sera.

The serum samples used in this study were referred to the clinicalbiology laboratory for routine screening or diagnosis of toxoplasmosis.These samples were tested with immuno fluorescence (IF) for IgG and IgM(BioM{acute over (e)}rieux, Brussels). The positive sera were givennumbers from 1 to 95 and the negative sera from 206 to 250. Those serawere tested again with the Toxo-IgG Micro ELA (ELISA) of BioM{acute over(e)}rieux. Two negative sera showing discrepant results with the IFAtest were eliminated from the panel. The reactivities obtained with thepanel of sera in the classical reference tests of Biom{acute over(e)}rieux are represented in table 2.

Parasites and lysate.

RH and Wiktor strains of Toxoplasma gondii were grown as described bySaavedra et al (1991).

Construction of c-DNA library in λgt11, screening and lysogenpreparation.

Described in Saavedra et al (1991).

Gel electrophoresis and western blotting. The total E. coli extracts orToxoplasma lysate were analyzed by SDS-PAGE (12.5%) in the presence ofβ-mercaptoethanol as described by Laemmli (1970). Eventually, proteinswere transferred to nitrocellulose membranes by the wet western blottingtechnique (Towbin et al., 1979) in carbonate buffer (10 mM NaHCO₃, 3 mMNa₂CO₃, 20% (v/v) methanol). The membrane was saturated with 5% fat freemilk in TNT (10 mM Tris, 150 mMNaCl, O,O5% (v/v)Tween20) for 1 h,followed by two washes in TNT. The membranes were incubated withmonoclonals or sera appropriately diluted in TNT containing 1% BSA for90 min. Before use, sera were preabsorbed on ice for 30 min using 10% E.coli lysate in the dilution buffer (TNT+1% BSA). After three washes withTNT, the bands were revealed with rabbit anti-mouse IgG conjugate (Dako,Denmark) or rabbit anti-human IgG conjugate (Dako, Denmark) AP labelled.Conjugates were diluted {fraction (1/2000)}. The AP activity wasrevealed by using the chromogenic substrate nitrobluetetrazolium-5-bromo-4-chloro-3-indolyl-phosphate in 50 mM trisHCl (pH9.5), 150 mM NaCl, 5 mM MgCl₂ buffer (Blake et al. 1984).

ELISA with recombinant antigens. ELISA plates (Immuno Plate MaxisorpF96; Nunc, Roskilde, Denmark) were coated by incubation at 37° C. for 1h. with a recombinant antigen solution (100 μl/well) in carbonate buffer0.1 M pH 9.5 for antigen Tg20 (2 μg/ml) or in glycine-HCL 0.2 M pH 4 forantigen Tg34AR (6 μ/ml). Blocking of the solid phase was carried out byincubation for 1 h at 37° with phosphate buffered saline (PBS)containing 0.1% casein (300 μl/well). After three washes with PBScontaining 0.05% Tween 20 (washing buffer), human sera were added at a{fraction (1/100)} dilution in sample diluent (PBS+0.1% casein+0.01%Triton X-705+1% E. coli lysate (v/V)). Incubation was done for 1.5 h at37° . The wells were then washed four times and incubated withhorseradish peroxidase-labelled goat anti-human IgG (Fc-fragment)Bethesda Research Laboratories, Gaitersburg, Md.) at a {fraction(1/5000)} dilution in PBS+0.1% casein, for 1 h at 37° . After fourwashes, the peroxidase activity was detected with H₂O₂ and3.3′,5,5′-tetramethyl-benzidine. The reaction was stopped after 30 minby adding 100 μl of 1 N H₂SO₄ and the O.D. was read at 450 nm.

Example 2: Identification and Sequencing of Clone Tg20

Antigen Tg34AR, a C-terminal fragment of Tg34 (=ROP2), detects 89% ofToxoplasma positive sera in ELISA as described by Van Gelder et al.1993. Antigen Tg34AR has been retested with the serum samples, describedin Example 1, resulting in a test sensitivity of 77% (cut off value=meanof O.D. of negative sera+3 standard deviations (sd), serum dilution{fraction (1/100)}).

Sera that were missed out by the Tg34AR ELISA, or that were onlyborderline positive, were used to screen a number of lambda gt11lysogens which had previously been described to be moderately reactivewith a pool of human and/or murine anti-T. gondii antibodies in WB(Saavedra et al.(1991)). This strategy leads to the identification ofclones which express an antigen “complementary” to Tg34AR, i.e. whichreacts with most of the sera which cannot be detected by the Tg34ARantigen.

The following λgt11 clones, belonging to different hybridisation groups,were chosen for screening: Tg6, Tg13, Tg18, Tg19, Tg20, Tg27, Tg34 andTg46. The lysogens of the clones were induced, producing β-galactosidase fusion proteins, and subsequently analyzed on Western Blotwith the above-selected sera.

Clones Tg13, Tg19 and Tg27 were probed with only three sera (#4, 21,33). They showed only a very weak reactivity and were therefore notanalysed further.

Clones Tg6, Tg18, Tg20, Tg46, Tg47 and Tg34 were probed with sera #4,21, 33, 17, 39, 53, 68, 45, 63, 79, 83 and 87. Serum #18 was used as apositive control (high titre in ELISA Tg34AR). Clone Tg34 is thepreviously characterised clone (=Rop2), of which Tg34AR is a fragment.

Clone Tg47 produced β galactosidase only, and was used as a negativecontrol.

Clone Tg20 reacted with {fraction (10/13)} sera, clone Tg6 reacted with{fraction (8/13)} sera, clone Tg46 reacted with {fraction (3/13)} sera.Sera #17, #45 and #63 did not react with any of the clones. From theseresults, clone Tg20 was chosen for further characterization andsequencing.

The EcoRI fragment of 1260 bp contained in clone Tg20 was transferredfrom λgt11 to pBluescript KS+, for restriction analysis and sequencing.To facilitate sequencing, one subclone containing the 5′ EcoRI-HindIIIfragment of 399 bp was ligated into pBluescript KS+ as well. Primerswere designed to elucidate the complete sequence of the clone. Everypart of the sequence was at least sequenced on both strands. Thesequence of the full EcoRI-insert (1260 bp) is shown in FIG. 1 (SEQ IDNO 1). An open reading frame of 705 bp (1-705) was identified, codingfor a protein with a theoretical calculated molecular weight of 25,695kDa, the sequence of which is represented in FIG. 1 (SEQ ID NO 2).

Example 3: Expression in E. coli of Recombinant Tg20 Antigen

1. Construction of recombinant vector

The 1260 bp by EcoRI-fragment of clone Tg20 was transferred to vectorpmTNFMPH (Innogenetics) for expression. This vector (see FIG. 2) enablesexpression of recombinant proteins in E. coli as fusion proteins with ashort mouse tumor necrosis factor (mTNF) peptide. Moreover, the vectoralso confers a polyhistidine sequence of six consecutive histidineresidues to the fusion protein, allowing fast and efficient purificationusing immobilized metal affinity chromatography (IMAC).

The vector pmTNFMPH was digested with ApaI and blunted with T4 DNApolymerase. The Tg20 1260 bp-fragment was recovered frompBluescriptKS+Tg20 by EcoRI digestion and blunting with T4 DNApolymerase. Ligation of both fragments with T4 DNA ligase confers an inframe fusion between the ORF of Tg20 and the mTNF-his6 leader peptide.

This fusion protein contains 37 aa provided by the leader peptide and235 aa encoded by the Toxoplasma gene fragment. The amino acid sequenceof the fusion protein is shown in FIG. 4 (SEQ ID NO 8).

2. E. coli expression of recombinant Tg20 antigen

The transcription of heterologous genes cloned in the expression vectorpmTNFMPH is initiated y the early leftward lambda promoter (P1) which iscontrolled by the C1 repressor. The host cell for expression is E. colistrain MC1061 [pAC1], containing a compatible plasmid which carries theC1-857 mutant gene, encoding a temperature sensitive variant of the C1repressor. This allows the initiation of expression of heterologousgenes by shifting the temperature from 28° C. to 42° C.

Cells of strain MC1061 [pAC1] were transformed with the expressionplasmid pmTNFMPH-Tg20 and grown at 28° C. An overnight culture was usedto inoculate ({fraction (1/100)}) 25 ml LB medium containing tetracyclin(10 μg/ml), which, was further grown at 28° C. (275 rpm) until the O.D.(at 600 nm) reached 0.2. The culture was divided into two equal parts,one of which was shifted to 42° C., while the second part was kept at28° C.

At 1.5 h intervals, samples were taken and analysed on SDS-PAGE andwestern blotting using anti-mTNF monoclonal antibodies. The fusionprotein mTNFTg20 was detectable on Coomassie Blue stained gels at a MWof approximately 29 kDa Western blots confirmed the identity of thisband, which was only present in the induced cultures. From theseexperiments the conditions for a large scale fermentation of the strainwere determined (see example 4).

3. Characterization of the Tg20 antigen: reaction with monoclonalantibodies and MW of the corresponding natural T. gondii antigen

Total lysate of cells expressing mTNF.H6Tg20 (shortly recombinant Tg20)was run on a polyacrylamide (PA) gel and transferred to a nitrocellulosemembrane. A set of monoclonal antibodies reacting with T. gondiiproteins with small molecular weight, as described by Saavedra et al(1990), were probed against with WB. Only monoclonal antibody BATO 214reacted with this antigen at a MW of about 29 kDa.

On a 12.5% PA gel total toxoplasma lysate and purified recombinant Tg20were run in parallel and transferred to nitrocellulose. The blot wasprobed with BATO 214 and revealed a MW for the naturally occurring T.gondii antigent of 24 kDa, while the recombinant antigen has a MW of 29kDa (see FIG. 5). The leader peptide of the recombinant antigen (mTNF)is about 2 kDa, which explains partly the larger size of the fusionprotein. In addition, when the sequence of Tg20 is analyzed for possibleeukaryotic cleavage sites (cleaving off signal peptides for secretion),three possible cleavage sites are found between aminoacid positions17-18, 20-21 and 25-26 (see arrows FIG. 1 b). Cleavage at one of thesepositions would reduce the MW of the recombinant to 24, 23.7 or 23.3kDa, in agreement with the MW of the natural T. gondii antigen. Thepresence of a cysteine residue in the possible signal peptide mayexplain the formation of a dimer of the fusion protein (see band atabout 58 kDa), which is absent in the naturally occurring (cleaved) Tg20protein.

Example 4. Purification of Recombinant Tg20 Antigen

A 151 fermentation was performed using an induction time of 3 h. Thecells were collected by low speed centrifugation and the pellet wasstored at −70° C. until further use.

One third of the cell pellet (9 g) was thawed, resuspended in 30 mllysis buffer (10 mM TrisHcl, 100 mM KCl, 5 mM EDTA. 25 mM ε-aminocaproicacid, 1 mM DTT, 1 mM PMSF) and passed twice through the French press.The lysate was centrifuged at 27000 g 30′, 4° C. The pellet obtained wasthen extracted with 20 ml 7 M guanidine chloride, 50 mM phosphate bufferpH7.2. The extract was centrifuged at 27000 g 30′, 4° C. The obtainedpellet was extracted again with 10 ml of the same buffer. Both extractswere pooled.

An IMAC column was prepared with chelating sepharose fast flow(Pharmacia), activated with NiCl₂ as described by the manufacturer, andequilibrated with 6 M guanidinium chloride 50 mM phosphate buffer pH7.2.

The pooled cell extract (30 ml) was loaded on the column. After washingwith running buffer (6 M guanidinium chloride, 50 mM phosphate bufferph7.2) a step elution was performed with 30, 60 and 100 mM imidazole inrunning buffer. Eluted fractions (6 ml) were analyzed on SDS-PAGE andWB. The fractions eluting at 100 mM imidazole contained the recombinantTg20 protein at a purification degree of about 95%. From 51 culture, 36mg purified protein in 29 ml was thus obtained (1.25 mg/ml) or 7.25 mg/lculture.

The purified recombinant protein showed a clear positive reaction in WBwith monoclonal antibodies anti-mTNF and BATO 214 and was furtherevaluated in ELISA with a panel of sera.

Example 5: Evaluation of Recombinant Tg20 in ELISA.

1. ELISA of Tg20 on G-bank sera.

Optimal conditions for coating Tg20 antigen on ELISA plates weredetermined as 2 μg/ml (100 μl/well) antigen and pH9.5 (carbonatebuffer). The serum dilution was {fraction (1/100)} and 1% E. coli lysatewas added to absorb anti- E. coli antibodies.

The serum panel (95 positive sera and 43 negative sera) were testedagainst Tg20 antigen (see table 2). The cutoff value (c.o.) wascalculated as the mean O.D. value (x) of the negative sera, plus 3standard deviations (s.d.), with the mean value being the sum of theindividual values divided by the number of values. Sensitivity andspecificity of the assay as compared to IF were 79% and 96%respectively. Furthermore, a correlation was shown between theImmunofluorescence (IF) titre and the sensitivity of the Tg20 ELISA.More Specifically, the Tg20 ELISA showed a sensitivity of 100% for serawith a IF-titre>{fraction (1/1024)}. For sera with an IF-titre of{fraction (1/256)}, sensitivity was 85%, while the sensitivity of theTg20 ELISA reached only 55% for sera with a IF-titre of {fraction(1/64)}.

2. ELISA of Tg34AR on G-bank sera

The serum samples of the G-bank (95 positive and 43 negative sera) werealso tested in ELISA Tg34AR, using the conditions as described by VanGelder et al. (1993) except for the serum dilution that is now {fraction(1/100)}. The results obtained are shown in table 2. Sensitivity andspecificity of the Tg34AR assay relative to the IF-test are 77% and 98%respectively. Again, the sensitivity was higher for sera with highIF-titre: sera with IF-titre>{fraction (1/1024)} were all positive inTg34AR ELISA (100% while sensitivity was only 89% and 41% for sera withIF-titres of respectively {fraction (1/256)} and {fraction (1/64)}.

3. Combination ELISA with Tg34AR and Tg20

Antigen Tg34AR is usually coated in glycine buffer pH4 (Saavedra et al1991). The optimal buffer for coating Tg20 antigen is carbonate pH9.5.Several buffers and pH were tested for both antigens, and from thisexperiment the best compromise for coating both antigens at the sametime was determined as carbonate buffer pH 9.5.

Both antigens were diluted in carbonate buffer and coated atconcentrations of 6 μg/ml for Tg34AR and 2 μg/ml for Tg20. Again 95positive and 43 negative sera with the G-bank were used to test theELISA. As described in Example 1, 1% E. coli lysate was added to thediluted serum ({fraction (1/100)}). The results obtained with theindividual sera are represented in Table 2. Sensitivity and specificityof this combination ELISA in comparison to the IF test are 92.5% and 98%respectively.

In conclusion, it can thus be said that antigent Tf34AR alone detects77% of the positive sera in this panel. Tg20 detects 79% of the positivesera. Both antigens combined detect 92.5% of the positive sera and thuspartially complement each other.

Example 6: Production of Deletion Clones of Tg20

In order to localize the most important epitopes of the Tg20 antigen,deletion clones were made, expressing only parts of the protein.

Clone Tg20 contained unique restriction sites at positions 585 (StyI),434 (NaeI) and 287 (Asp700) FIG. 1 a). The fragments were cut out ofpmTNFMPH.Tg20 by digestion with BamHI-StyI (StyI site blunted,BamHI-NaeI and BamHI-Asp700 respectively. The fragments were ligatedinto vector pIGFH111 or pIGFH10 digested with BamHI-StuI.

The vectors pIGFH111 or pIGFH10 digested with BamHI-StuI.

The vectors pIGFH111 (see FIG. 3) and pIGFH10 (Innogenetics N.V.) arederivatives of pMTNFMPH and have the same basic properties. They containa larger number of unique restriction sites in the polylinker sequenceand the ε-enhancer sequence has been inserted in order to give higherexpression levels. Plasmid pIGFH10 is the same as pIGHF111 except forthe ApaI unique restriction site in the polylinker sequence which ismissing in the former.

The principle and protocol to obtain expression of a heterologous genein pIGFH111 (and pIGFH10 ) is the same as for pmTNFMPH (described inExample 3). Strain SG4044 [pACI] was used. Three different clones,corresponding to the three different restriction fragments, were testedfor expression of the corresponding antigent fragments: Tg20BS(BamHI-StyI), Tg20BN (BamHI - NaeI) and Tg20BA (BamHI - Asp700).

The samples collected from the induction experiments were analysed onWestern blots and probed with anti-mouse TNF monoclonal and with BATO214. All deletion clones reacted with anti-mouse TNF at the expectedsize, i.e. Tg20BS at approximately 25 kDa, Tg20BN at approximately 20kDa (with a major degradation band around 15 kDa) and Tg20BA atapproximately 14 kDa. Deletion clone Tg20BS gave only very lowexpression levels. For Tg20BN a lot of “leak” expression was observed,i.e. expression at the non-permissive temperature of 28° C.

When probed with BATO 214 only the two largest deletion clones, i.e.Tg20BS and Tg20BN reacted. This gives a good indication for the positionof the BATO 214 epitope on antigen Tg20, i.e. between amino acidposition 95 (bp 287) and amino acid position 145 (bp 434) on FIG. 1 b.Testing with human sera indicates that the main epitopes important inthe recognition of human sera are also located in this region (seeexample 7 and 8).

Clones Tg20BN and Tg20BA were further purified and evaluated in ELISA(see examples 7 and 8). The expression level of clone Tg20BS was too lowfor purification purposed.

Example 7: Purification of Tg20BN and Evaluation in ELISA

1. Purification of Tg20BN antigent fragment

Tg20BN was purified on IMAC as described above for Tg20 purification,with some modifications. A 151 fermentation was carried out, but growthand expression were carried out at 28° C. only. The “leak” expressionlevel at 28° C. was sufficient for purification purposes and it preventsthe more pronounced degradation of the recombinant protein seen at theincreased temperature of 42° C. After French press lysis of one cellpellet (51) in lysis buffer (10 mM sodium phosphate, 100 mM KCl, 10 mMε-amino caproic acid), 2 mM PMSF was added. The lysate was centrifugedand the soluble fraction was used for purification.

Guanidinium chloride powder was added to reach a final concentration of6 M and the solution was centrifuged again. Imidazole was added to reacha concentration of 20 mM. Starting the purification with 20 mM imidazoleprevents some E. coli contaminants to bind to the column.

An IMAC column was made with chelating sepharose fast flow (Pharmacia),activated with niCl₂ as described by the manufacterer, and equilibratedwith running buffer (6 M guanidine chloride, 50 mM phosphate, 20 mMimidazole, pH 7.2).

The sample (50 ml) was loaded and after washing with running buffer, astep elution was performed with 35, 50 and 200 mM imidazole in runningbuffer. Eluted fractions (3 ml) were analysed on WB and SDS-PAGE. The200 mM fractions contained the recombinant purified protein, that wassufficiently pure (95%). Three fractions (=9 ml) were pooled, the poolcontaining 230 μg/ml Tg20BN.

2. ELISA with Tg20BN

ELISA plates were coated with antigen Tg20 and Tg20BN separately at 2μg/ml in carbonate buffer. To the sample diluent 1% E. coli lysate wasadded and the serum was diluted {fraction (1/100)}.

Of the G-bank 50 positive (#1 to #50) and 37 negative (#206 to #245except #221,#228,#233) sera were tested and the results for Tg20 andTg20BN were compared (see table 3). At a cutoff value of (mean+3 s.d.)no false positives were detected with either test (100% specificity). Asto the sensitivity of both assays, the ELISA with the full Tg20 antigenresulted in a sensitivity of 90%, while the truncated protein Tg20BNyielded a sensitivity of 78%.

Most of the sera that are missed by Tg20BN-ELISA are only borderlinepositive with Tg20-ELISA (see table 3), except for serum #35 and #40which show a clear drop in reactivity when tested with the deletionclone Tg20BN.

Since the large majority of the sera are still detected by Tg20BNantigen, it can be concluded that the immunodominant epitopes arelocated on the N-terminal part (aa 1 to aa 145) of the Tg20 antigen. Fora few sera (like #35 and #40) an epitope on the C-terminal part seems tobe important. These two sera are thus missed by the Tg20BN ELISA, but itis important to note that they are detected by the Tg34AR ELISA (cfr.Table 2).

Example 8: Purification of Tg20BA and Evaluation in ELISA

1. Purification of Tg20BA

The IMAC-purification of Tg20BA was carried out on small scale withNi-NTA spin columns (Qiagen). A culture of 100 ml MC1061 [pACI]transformed with pIGFH10Tg20BA was induced (42° C.) for 2 h. Cells wereharvested and lysed in 6 M guanidinium.HCl, 50 mM phosphate pH 8.3(shaking for 45 min.). After centrifugation a cleared lysate wasobtained and this was loaded on the spin column after the column wasequilibrated with the same buffer. The column was washed twice with 6 MguanidiniumHCl, 50 mM phosphate buffer at pH 6.3. Elution was done with6 M guanidinium.HCl, 50 mM phosphate pH 4.3. This elution step wasrepeated. The eluates were checked on WB. The first eluate contains mostof the purified recombinant protein and is 95% pure. It contained 265μg/ml in 600 μl.

2. ELISA with Tg20BA

From the G-bank 31 sera that gave a positive reaction with Tg20BN weretested on the smaller fragment, Tg20BA, including also 8 negative sera.Tg20BA gave no reaction higher than the cutoff value with these sera(results now shown). This indicates that the Tg20BA fragment (aa pos. 1to aa pos. 95) does not contain any epitopes important for therecognition of human sera from infected individuals.

From the differential reactivity of antigen fragment Tg20BN and Tg20BAit can be concluded that the main epitope(s) important in therecognition of the humoral immune response are localized in the fragmentextending from aa pos. 95 to aa pos. 145, or at least that this regionis important for the construction of an immunoreactive epitope.

Further fine mapping of the epitope(s) can be performed using anoverlapping set of synthetic peptides.

Example 9: The Combination of Tg34AR and Tg20BN in ELISA

Antigen fragment Tg20 BN was combined in ELISA with Tg34AR to comparethe results of this combination ELISA with the results obtained earlierwith the combination of Tg20 and Tg34AR (see Table 2). Again, thecoating concentration of Tg34AR was 6 μg/ml and for Tg20BN aconcentration of 2 μg/ml was used. The sensitivity of the combinationELISA with the full size antigen Tg20 (Tg34AR) was only slightly higher(92.5%) as compared to the combination ELISA of the antigen fragmentTg20BN with Tg34AR (sensitivity 91.5%). From this it can be concludedthat the most important B-cell epitopes of the Tg20 antigen are locatedin the Tg20BN-fragment.

Example 10: Localisation of the Dominant Human Epitope on Tg20BN withPeptides

In order to localize the most important B-cell epitope(s) more preciselyin the Tg20-BN fragment, a series of overlapping peptides aresynthezised, covering the sequence region extending from aminoacidposition 95 to amino acid position 145 in FIG. 1 b. A biotin moleculewas added at the N-terminus during synthesis. The peptides have anoverlap of 10 amino acids, and are represented by the followingsequences:

GluProAspGluGlnGluGluValHisPheArgLysArgGlyValArgSerAspAlaGlu (SEQ ID NO3, aa90-110)

ArgLysArgGlyValArgSerAspAlaGluValThrAspAspAsnIleTyrGluGluHis (SEQ ID NO4, aa100-120)

ValThrAspAspAsnIleTyrGluGluHisThrAspArgLysValValProArgLysSer (SEQ ID NO5, aa110-130)

ThrAspArgLysValValProArgLysSerGluGlyLysArgSerPheLysAspLeuLeu (SEQ ID NO6, aa120-140)

GluGlyLysArgSerpHeLysAspLeuLeuLysLysLeuAlaLeuPro (SEQ ID NO 7,aa130-146)

Microtiter plates are coated with streptavidin by incubating 100 μl/wellof a 5 μg/ml streptavidin solution in carbonate buffer (50 mM, pH 9.6)for 1 h at 37° C. After washing, the biotinylated peptides are added tothe wells (100 ng/well, each well (in duplicate) representing adifferent peptide. Peptides are let to bind the streptavidin for 1 h at37° C. after which washing occurs. The ELISA procedure is then furthercontinued as described for Tg20 ELISA in Example 1. The five peptideswere tested with two positive human sera (18,23), one negative serum(210) and with mouse monoclonal BATO 214 and an unrelated monoclonal(BATO 35). Both monoclonal preparations were dilutions ({fraction(1/2000)}) from ascites produced in mice. The conjugate used in theELISA with the monoclonals was rabbit anti-mouse conjugate (Sigma,dilution {fraction (1/5000)}). The results with the human sera do notindicate one peptide as the more important, all petides react to agreater or smaller extent. Monoclonal BATO 214 reacts with petides SEQID NO 3 and SEQ ID NO 4. This probably defines the epitope for BATO 214between aa 100 and 110.

peptide serum aa90-110 aa100-120 aa110-130 aa120-140 aa130-146 18 .645.646 .840 .505 .400 23 .517 .463 .595 .764 .919 210  .120 .120 .143 .182.212 BATO214 1.350   .2169 .007 .009 .007 BATO35 .010 .009 .010 .010.010

Those peptides which seem to contain an important epitope, are furthermapped using a set of smaller overlapping peptides (8-mers) tested inthe same way as described above.

REFERENCES

Asseline U. Delarue M, Lancelot G, Toulme F, Thuong N (1984) Nucleicacid-binding molecules with high affinity and base sequencespecificity:intercalating agents covalently linked tooligodeoxynucleotides. Proc. Natl. Acad. Sci. USA 81(11):3297-301.

Blake M S, Johnston K H, Russell-Jones G J, Gotschlich E C (1984). Arapid, sensitive method for detection of alkaline phosphatase-conjugatedanti-antibody on Western blots. Anal. Biochem. 136, 175-179.

Beverly J K A (1976). Toxoplasmosis in animals. Vet. Rec. 99, 123-127.

Blalock J (1990) Complementarity of peptides specified by ‘sense’ and‘antisense’ strands of DNA. Trends Biotechnol. 8: 140-144.

Burg J L, Perelman D, Kasper L H; Ware P L, Boothroyd J C (1988).Molecular analysis of the gene encoding the major surface antigen ofToxoplasma gondii. J. Immunol. 141, 3584-3591.

Buxton D, Thomson K M, Maley S. Wright S, Box H J (1991). Vaccination ofsheep with a live incomplete strain (S48) of Toxoplasma gondii and theirimmunity to challenge when pregnant. Vet. rec. 192, 89-93.

Buxton D, Thomson K M. Maley S. Wright S, Box H J (1993). Experimentalchallenge of sheep 18 months after vaccination with a live (S48)Toxoplasma gondii vaccine. Vet. rec. 133, 310-312.

Cesbron-Delauw M F, Guy B, Torpier G, Pierce R J, Lenzen G, Cesbron J Y,Charif H, Lepage P, Darcy F, Lecocq J P, Capron, A. (1989). Molecularcharacterisation of a 23-kilodalton major antigen secreted by Toxoplasmagondii. Proc. Natl. Acad. Sci. USA 86, 7537--7541.

Compton J (1991). Nucleic acid sequence-based amplification. Nature,350: 91-92.

de Pr{acute over (e)}val (1978) Immunoglobulins, In: Bach J Immunology,New York, Wiley and Sons: 144-219.

Duck P (1990). Probe amplifier system based on chimeric cyclingoligonucleotides. Biotechniques 9, 142-147.

Fleishmann J, Davie J (1984) Immunoglobulins: allotypes and idiotypes.In: Paul W (Ed) Fundamental Immunology, New York, Raven Press: 205-220.

Frenkel J K (1967). Adoptive immunity to intracellular infection. J.Immunol. 98, 1309-1319.

Gazinelli R T, Hakim F T, Hieny S, Shearer G M, Sher A (1991).Synergistic role of CD4+and CD8+T lymphocytes in IFN-y production andprotective immunity induced by an attenuated Toxoplasma gondii vaccine.J. Immunol. 146, 286-292.

Gheuens J, Mc Farlin D (1982) Use of monoclonal anti-idiotypic antibodyto P3-X6Ag8 myeloma protein for analysis and purification of Blymphocyte hybridoma products. Eur J Immunol 12: 701-703.

Chiso J, Saball E, Leoni J, Rostagno A, Frangio (1990) Binding ofcystatin C to C4: the importance of antisense peptides and theirinteraction. Proc Natl Acad Sci (USA) 87: 1288-1291.

Guatelli J, Whitfield K, Kwoh D, Barringer K, Richman D, Gengeras T(1990) Isothermal, in vitro amplification of nucleic acids by amultienzyme reaction modeled after retroviral replication. Proc NatlAcad Sci USA 87: 1874-1878.

Hughes H P A (1985). Toxoplasmosis: the need for improved diagnostictechniques and accurate risk assessment. Curr. Top. Microbiol. Immunol.120, 105-139.

Jacobs K, Rudersdorf R, Neill S, Dougherty J, Brown E, Fritsch E (1988).The thermal stability of oligonucleotide duplexes is sequenceindependent in tetraalkylammoniuim salt solution: application toidentifying recombinant DNA clones. Nucl Acids Res 16:4637-4650.

Kwoh D, Davis G, Whitfield K, Chappelle H, Dimichele L, Gingeras T(1989). Transcription-based amplification system and detection ofamplified human immunodeficiency virus type 1 with a bead-based sandwichhybridization format. Proc Natl Acad Sci USA, 86: 1173-1177.

Kwok S, Kellogg D, McKinney N, Spasic D, Goda L, Levenson C, Sinisky J,(1990). Effects of primer-template mismatches on the polymerase chainreaction: Human immunodeficiency view type 1 model studies. Nucl. AcidsRes., 18:999.

Laemmli U K (1970). Cleavage of structural proteins during the assemblyof the head of bacteriophage T4. Nature 227, 680-685.

Landgren U, Kaiser R, Sanders J, Hood L (1988). A ligase-mediated genedetection technique. Science 241:1077-1080.

Lecordier L, Moleon-Borodowsky I, Dubremetz J-F, Tourvielle B, MercierC, Desl{acute over (e)}e D, Capron A, Cesbron-Delauw M-F (1995).Characterisation of a dense granule antigen of Toxoplasma gondii (GRA6)associated to the network of the parasitophorous vacuole. Mol. Biochem.Parasitol. 70, 85-95.

Lomeli H, Tyagi S, Printchard C, Lisardi P, Kramer F (1989) Quantitativeassays based on the use of replicatable hybridization probes. Clin Chem35: 1826-1831.

McLeod R, Frenkel J K, Estes R G, Mack D G, Eisenhauer P B, Gibori G(1988). Subcutaneous and intestinal vaccination with tachyzoites ofToxoplasma gondii and acquisition of immunity to peroral and congenitalToxoplasma challenge. J. Immunol. 140, 1632-1637.

Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning. Alaboratory manuel. Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.

Matsukura M, Shinozuka K, Zon G, Mitsuya H, Reitz M, Cohen J, Broder S(1987) Phosphorothioate analogs of oligodeoxynucleotides: inhibitors ofreplication and cytopathic effects of human immunodeficiency virus.Proc. Natl. Acad. Sci. USA 84(21):7706-10.

Mercier C, Lecordier L, Darcy F, Deslee D, Murray A, Tourvieille B, MaesP, Capron A, Cesbron-Delauw M-F (1993). Molecular characterisation of adense granule antigen (Gra 2) associated with the parasitophorus vacuolein Toxoplasma gondii. Mol. Biochem. Parasitol. 58, 71-82.

Miller P, Yano J, Yano E, Carroll C, Jayaram K, Ts'o P (1979) Nonionicnucleic acid analogues. Synthesis and characterization ofdideoxyribonucleoside methylphosphonates. Biochemistry 18(23): 5134-43.

Nathan C F, Predergast F J, Wiebe M E, Stanley E R, Platze E, Remold HG, Welte K, Rubin B Y and Murray H W (1984). Activation of humanmacrophages: comparison of other cytokines with interferon-gamma. J.Exp. Med. 160, 600-605.

Neilsen P, Egholm M, Berg R, Buchardt O (1991) Sequence-selectiverecognition of DNA by strand displacement with a thymine-substitutedpolyamide. Science 254(5037): 1497-500.

Neilsen P, Egholm M, Berg R, Buchardt O (1993) Sequence specificinhibition of DNA restriction enzyme cleavage by PNA. Nucleic-Acids-Res.21(2): 197-200.

Parmley S F, Sgarlato G D, Mark J, Prince J B, Remington J S (1992).Expression, characterisation, and serologic reactivity of recombinantsurface antigen P22 of Toxoplasma gondii. J. Clin. Microbiol. 30,1127-1133.

Parmley S F, Sgarlato G D, Remington J S (1993). Genomic and correctedcDNA sequence of the P28 gene from Toxoplasma gondii. 57, 161-165.

Perrson M, Caothien R, Burton D (1991) Generation of diverse highaffinity human monoclonal antibodies by repertoire cloning. Proc. Natl.Acad. Sci. 88:2432-2436.

Pfefferkorn E R (1984). Interferon gamma blocks the growth of Toxoplasmagondii in human fibroblasts by inducing the host cells to degradetryptophan. Proc. Natl. Acad. Sci. USA 81, 908-912.

Prince J B, Araujo F G, Remington J S, Burg J L, Boothroyd J C, Sharma SD (1989). Cloning of cDNAs encoding a 28 kilodalton antigen ofToxoplasma gondii. Mol. Biochem. Parasitol. 34, 3-14.

Remaut E. Tsao H, Fiers W (1983). Improved plasmid vectors with athermoinducible expression and temperature-regulated runawayreplication. Gene 22, 103-113.

Remington J. S. and Krahenbuhl, J. L. (1982) in Immunology of humaninfection, eds. Nahmias, A. J. and O'Reilly, R. J. (Plenum Publishing,New York), Part II, pp. 327-372.

Roubos E (1990) Sense-antisense complementarity of hormone receptorinteraction sites. Trends Biotechnol 8: 279-281.

Saavedra R, De Meuter F, Decourt J-L, H{acute over (e)}rion P, (1991).Human T cell clone identifies a potentially protective 54-kDA proteinantigen of Toxoplasma gondii cloned and expressed in Escherichia coli.J. Immunol. 147, 1975-1982.

Saavedra R, De Meuter F, H{acute over (e)}rion P, (1990). Monoclonalantibodies identify new Toxoplasma gondii soluble antigens. Hybridoma 9,453-463.

Saiki R, Gelfand D, Stoffel S, Scharf S, Higuchi R, Horn G, Mullis K,Erlich H (1988). Primer-directed enzymatic amplification of DNA with athermostable DNA polymerase. Science 239:487-491.

Sethi K K, Omata Y, Brandis H, (1985). Contribution of immune interferon(INF-gamma) in lymphokine-induced anti-Toxoplasma activity: studies withrecombinant murine IFN-gamma. Immunobiology 170, 270-283.

Sibley L D, Pfefferkorn E R, Boothroyd J C (1991). Proposal for auniform genetic nomenclature in Toxoplasma gondii. Parasitology Today 7,327-328.

Suzuki Y, Orellana M A, Schreiber R D, Remington J S (1988).Interferon-gamma: the major mediator of resistence against Toxoplasmagondii. Science 240, 516-518.

Suzuki Y, Remington J S (1988). Dual regulation of resistance againstToxoplasma gondii infection by Lyt-2⁺ and Lyt-1⁻³⁰ ,L3T4⁺T cells inmice. J. Immunol. 140, 3943-3946.

Suzuki Y, Remington J S (1990). The effect of anti-IFN-gamma antibody onthe protective effect of Lyt-2⁺ immune T cells against toxoplasmosis inmice. J. Immunol. 144, 1954-1956.

Towbin H, Staehelin T, Gordon J (1979). Eletrophoretic transfer ofproteins from polyacrylamide gels to nitrocellulose sheets: procedureand some applications. Proc. Natl. Acad. Sci. USA. 76, 4350-4354.

Van Gelder P, Bosman F, De Meuter F, van Heuverswyn H, and H{acute over(e)}rion P (1993). Serodiagnosis of toxoplasmosis by using a recombinantform of the 54- dilodalton rhoptry antigen expressed in Escherichiacoli. J. Clin Microbiol. 31, 9-15.

Waldeland H, Frenkel J K (1983). Live and killed vaccines againsttoxoplasmosis in mice. J. Parasitol. 69, 60.

Walker G, Little M, Nadeau J, Shank D (1992). Isothermal in vitroamplification of DNA by a restriction enzyme/DNA polymerase system. ProcNatl Acad Sci USA 89:392-396.

Wu D, Wallace B (1989). The ligation amplification reaction(LAR)—amplification of specific DNA sequences using sequential rounds oftemplate-dependent ligation. Genomics 4:560-569. Barany F (1991).Genetic disease detection and DNA amplification using clonedthermostable ligase. Proc Natl Acad Sci USA 88:189-193.

8 1260 base pairs nucleic acid single linear DNA (genomic) unknown CDS1..708 mat_peptide 1..705 1 GAA TTC GGC GCA ATT TTT TCC GCG CTT TGT GTTTTA GGC CTG GTG GCG 48 Glu Phe Gly Ala Ile Phe Ser Ala Leu Cys Val LeuGly Leu Val Ala 1 5 10 15 GCG GCT TTG CCC CAG TTC GCT ACC GCG GCC ACCGCG TCA GAT GAC GAA 96 Ala Ala Leu Pro Gln Phe Ala Thr Ala Ala Thr AlaSer Asp Asp Glu 20 25 30 CTG ATG AGT CGA ATC CGA AAT TCT GAC TTT TTC GATGGT CAA GCA CCC 144 Leu Met Ser Arg Ile Arg Asn Ser Asp Phe Phe Asp GlyGln Ala Pro 35 40 45 GTT GAC AGT CTC AGA CCG ACG AAC GCC GGT GTC GAC TCGAAA GGG ACC 192 Val Asp Ser Leu Arg Pro Thr Asn Ala Gly Val Asp Ser LysGly Thr 50 55 60 GAC GAT CAC CTC ACC ACC AGC ATG GAT AAG GCA TCT GTA GAGAGT CAG 240 Asp Asp His Leu Thr Thr Ser Met Asp Lys Ala Ser Val Glu SerGln 65 70 75 80 CTT CCG AGA AGA GAG CCA TTG GAG ACG GAG CCA GAT GAA CAAGAA GAA 288 Leu Pro Arg Arg Glu Pro Leu Glu Thr Glu Pro Asp Glu Gln GluGlu 85 90 95 GTT CAT TTC AGG AAG CGA GGC GTC CGT TCC GAC GCT GAA GTG ACTGAC 336 Val His Phe Arg Lys Arg Gly Val Arg Ser Asp Ala Glu Val Thr Asp100 105 110 GAC AAC ATC TAC GAG GAG CAC ACT GAT CGT AAG GTG GTT CCG AGGAAG 384 Asp Asn Ile Tyr Glu Glu His Thr Asp Arg Lys Val Val Pro Arg Lys115 120 125 TCG GAG GGC AAG CGA AGC TTC AAA GAC TTG CTG AAG AAG CTC GCGCTG 432 Ser Glu Gly Lys Arg Ser Phe Lys Asp Leu Leu Lys Lys Leu Ala Leu130 135 140 CCG GCT GTT GGT ATG GGT GCA TCG TAT TTT GCC GCT GAT AGA CTTGTG 480 Pro Ala Val Gly Met Gly Ala Ser Tyr Phe Ala Ala Asp Arg Leu Val145 150 155 160 CCG GAA CTA ACA GAG GAG CAA CAG AGA GGC GAC GAA CCC CTAACC ACC 528 Pro Glu Leu Thr Glu Glu Gln Gln Arg Gly Asp Glu Pro Leu ThrThr 165 170 175 GGC CAG AAT GTG GGC ACT GTG TTA GGC TTC GCA GCG CTT GCTGCT GCC 576 Gly Gln Asn Val Gly Thr Val Leu Gly Phe Ala Ala Leu Ala AlaAla 180 185 190 GCA GCG TTC CTT GGC ATG GGT CTC ACG AGG ACG TAC CGA CATTTT TCC 624 Ala Ala Phe Leu Gly Met Gly Leu Thr Arg Thr Tyr Arg His PheSer 195 200 205 CCA CGC AAA AAC AGA TCA CGG CAG CCT GCA CTC GAG CAA GAGGTG CCT 672 Pro Arg Lys Asn Arg Ser Arg Gln Pro Ala Leu Glu Gln Glu ValPro 210 215 220 GAA TCA GGC GAA GAT GGG GAG GAT GCC CGC CAG TAGGATATGGGGGCTAATAA 725 Glu Ser Gly Glu Asp Gly Glu Asp Ala Arg Gln 225 230 235AAGTGAGTAG GAGCTCGAGG ACAGTGTCCC GAACGCGCCT GAGAGGCAGA CAGACACAGA 785AGAGTGAAGA AAAACAACAT GGTATTACGT GCGGTGAGTG TTTGCTGTCA CGTGTTTTTT 845GCGCCACAAA GACAGCTTGT GTTGTATGCA TGGGATCGAC AGTTCATGGA CGGCGCTACC 905CAGAGAGGCG GCATTTGCGT ACACCGTGGG TCGTCATGAG TACCGGGACA TCGTGTTCGT 965GTTTATTTGT TCATGTCGAA GTGCACTAAG ACACGAGACG AAAGGGTGGT TCCGCCCCTG 1025GCAGCATCAC GTAGTGGTTT CTTTGTCGAG AACAGCGGCA GTCCGAGGCC ACTTGAGACA 1085GGATGTTTGA GTGTATACAG ACAACGTGGT CACAGCATGA GGCAAAGCTG TCTAAGCAGC 1145CATTTGCGCG AGCGAAGTCA TCCATGCCGA CTGTGTGAGC CTCTTTCGTC ACTTTGAATG 1205AGACAGAAAC TAAGACTCGC AGCAGGTCTG AATATTGCGA ATAAAAACCG AATTC 1260 235amino acids amino acid linear protein unknown 2 Glu Phe Gly Ala Ile PheSer Ala Leu Cys Val Leu Gly Leu Val Ala 1 5 10 15 Ala Ala Leu Pro GlnPhe Ala Thr Ala Ala Thr Ala Ser Asp Asp Glu 20 25 30 Leu Met Ser Arg IleArg Asn Ser Asp Phe Phe Asp Gly Gln Ala Pro 35 40 45 Val Asp Ser Leu ArgPro Thr Asn Ala Gly Val Asp Ser Lys Gly Thr 50 55 60 Asp Asp His Leu ThrThr Ser Met Asp Lys Ala Ser Val Glu Ser Gln 65 70 75 80 Leu Pro Arg ArgGlu Pro Leu Glu Thr Glu Pro Asp Glu Gln Glu Glu 85 90 95 Val His Phe ArgLys Arg Gly Val Arg Ser Asp Ala Glu Val Thr Asp 100 105 110 Asp Asn IleTyr Glu Glu His Thr Asp Arg Lys Val Val Pro Arg Lys 115 120 125 Ser GluGly Lys Arg Ser Phe Lys Asp Leu Leu Lys Lys Leu Ala Leu 130 135 140 ProAla Val Gly Met Gly Ala Ser Tyr Phe Ala Ala Asp Arg Leu Val 145 150 155160 Pro Glu Leu Thr Glu Glu Gln Gln Arg Gly Asp Glu Pro Leu Thr Thr 165170 175 Gly Gln Asn Val Gly Thr Val Leu Gly Phe Ala Ala Leu Ala Ala Ala180 185 190 Ala Ala Phe Leu Gly Met Gly Leu Thr Arg Thr Tyr Arg His PheSer 195 200 205 Pro Arg Lys Asn Arg Ser Arg Gln Pro Ala Leu Glu Gln GluVal Pro 210 215 220 Glu Ser Gly Glu Asp Gly Glu Asp Ala Arg Gln 225 230235 20 amino acids amino acid single linear peptide unknown 3 Glu ProAsp Glu Gln Glu Glu Val His Phe Arg Lys Arg Gly Val Arg 1 5 10 15 SerAsp Ala Glu 20 20 amino acids amino acid single linear peptide unknown 4Arg Lys Arg Gly Val Arg Ser Asp Ala Glu Val Thr Asp Asp Asn Ile 1 5 1015 Tyr Glu Glu His 20 20 amino acids amino acid single linear peptideunknown 5 Val Thr Asp Asp Asn Ile Tyr Glu Glu His Thr Asp Arg Lys ValVal 1 5 10 15 Pro Arg Lys Ser 20 20 amino acids amino acid single linearpeptide unknown 6 Thr Asp Arg Lys Val Val Pro Arg Lys Ser Glu Gly LysArg Ser Phe 1 5 10 15 Lys Asp Leu Leu 20 16 amino acids amino acidsingle linear peptide unknown 7 Glu Gly Lys Arg Ser Phe Lys Asp Leu LeuLys Lys Leu Ala Leu Pro 1 5 10 15 272 amino acids amino acid singlelinear protein unknown 8 Met Val Arg Ser Ser Ser Gln Asn Ser Ser Asp LysPro Val Ala His 1 5 10 15 Val Val Ala Asn His Gln Val Glu Glu Gln GlyIle His His His His 20 25 30 His His Val Asp Pro Glu Phe Gly Ala Ile PheSer Ala Leu Cys Val 35 40 45 Leu Gly Leu Val Ala Ala Ala Leu Pro Gln PheAla Thr Ala Ala Thr 50 55 60 Ala Ser Asp Asp Glu Leu Met Ser Arg Ile ArgAsn Ser Asp Phe Phe 65 70 75 80 Asp Gly Gln Ala Pro Val Asp Ser Leu ArgPro Thr Asn Ala Gly Val 85 90 95 Asp Ser Lys Gly Thr Asp Asp His Leu ThrThr Ser Met Asp Lys Ala 100 105 110 Ser Val Glu Ser Gln Leu Pro Arg ArgGlu Pro Leu Glu Thr Glu Pro 115 120 125 Asp Glu Gln Glu Glu Val His PheArg Lys Arg Gly Val Arg Ser Asp 130 135 140 Ala Glu Val Thr Asp Asp AsnIle Tyr Glu Glu His Thr Asp Arg Lys 145 150 155 160 Val Val Pro Arg LysSer Glu Gly Lys Arg Ser Phe Lys Asp Leu Leu 165 170 175 Lys Lys Leu AlaLeu Pro Ala Val Gly Met Gly Ala Ser Tyr Phe Ala 180 185 190 Ala Asp ArgLeu Val Pro Glu Leu Thr Glu Glu Gln Gln Arg Gly Asp 195 200 205 Glu ProLeu Thr Thr Gly Gln Asn Val Gly Thr Val Leu Gly Phe Ala 210 215 220 AlaLeu Ala Ala Ala Ala Ala Phe Leu Gly Met Gly Leu Thr Arg Thr 225 230 235240 Tyr Arg His Phe Ser Pro Arg Lys Asn Arg Ser Arg Gln Pro Ala Leu 245250 255 Glu Gln Glu Val Pro Glu Ser Gly Glu Asp Gly Glu Asp Ala Arg Gln260 265 270

What is claimed is:
 1. A purified and isolated polypeptide or peptidecontaining a sequence selected from the group consisting of: (1) anamino acid sequence extending from an amino acid position x to an aminoacid position y in the sequence SEQ ID NO 2, wherein x=1 and y=196, orx=1 and y=160, or x=1 and y=145, or x=95 and y=145, (2) a fragment of anamino acid sequence of (1), said fragment comprising at least 7contiguous amino acids from amino acid 96 through amino acid 196 of SEQID NO:2, and (3) an equivalent of an amino acid sequence of (1) or afragment of (2) wherein said equivalent comprises said amino acidsequence of (1) or said fragment of (2) wherein at least one amino acidof said sequence or fragment has been replaced by an amino acid as shownin Table 1, said polypeptide or peptide containing a reactive epitopewhich produces at least one of a humoral and a cell-mediated response ina T. gondii infected host organism.
 2. A purified and isolatedpolypeptide or peptide containing a sequence selected from the groupconsisting of: (1) the amino acid sequence extending from an amino acidposition 95 to an amino acid position 145 of SEQ ID NO 2, (2) a fragmentof the amino acid sequence of (1) comprising at least 7 contiguous aminoacids from amino acid 96 through amino acid 145 of SEQ ID NO:2, (3) atleast one of the amino acid sequences represented byGluProAspGluGlnGluGluValHisPheArgLysArgGlyValArgSerAspAlaGlu (SEQ ID NO3), or ArgLysArgGlyValArgSerAspAlaGluValThrAspAspAsnIleTyrGluGluHis (SEQID NO 4), orValThrAspAspAsnIleTyrGluGluHisThrAspArgLysValValProArgLysSer (SEQ ID NO5), or ThrAspArgLysValValProArgLysSerGluGlyLysArgSerPheLysAspLeuLeu (SEQID NO 6), or GluGlyLysArgSerPheLysAspLeuLeuLysLysLeuAlaLeuPro (SEQ ID NO7), and (4) an equivalent of an amino acid sequence of (1), a fragmentof (2) or at least one of the amino acid sequences of (3) wherein saidequivalent comprises said amino acid sequence of (1) or said fragment of(2) or said amino acid sequence of (3) wherein at least one amino acidof said sequence or fragment has been replaced by an amino acid as shownin Table 1, said polypeptide or peptide containing a reactive epitopewhich produces at least one of a humoral and a cell-mediated immuneresponse in a T. gondii infected host organism.
 3. A fusion proteinconsisting of a polypeptide or fragment thereof according to claim 1linked to a heterologous polypeptide sequence.
 4. A method for detectingantibodies to T. gondii in a sample, comprising: contacting said samplewith at least one polypeptide or peptide according to claim 1 underconditions sufficient to form an immunological complex between saidpolypeptide or peptide and said antibodies, if present, and, detectingsaid immunological complex.
 5. A method according to claim 4 whereinsaid sample is a biological sample from an individual and said methodfurther comprises contacting said sample with at least one further T.gondii polypeptide or peptide for measuring the cellular immune responseof said individual.
 6. A kit for detecting anti-T. gondii antibodies ina sample, said kit comprising: at least one polypeptide or peptideaccording to claim 1, optionally in combination with other polypeptidesor peptides being also optionally immobilized of a solid support,optionally, a buffer, or components necessary for producing the buffer,enabling a binding reaction to occur between the antibodies present insaid sample and said polypeptides or peptides, optionally, a means fordetecting the immune complex formed, and, optionally, an automatedscanning and interpretation device for inferring the presence of saidantibodies in said sample.
 7. A kit according to claim 6, furthercomprising at least one non-T. gondii pathogen-specific detectionreactant selected from the group consisting of a non-T. gondii,pathogen-specific, polypeptide, antibody and polynucleic acid.
 8. Apurified and isolated polypeptide or peptide according to claim 1containing a sequence selected from the group consisting of: (1) anamino acid sequence extending from an amino acid position x to an aminoacid position y in the sequence SEQ ID NO:2, wherein x=1 and y=196, orx=1 and y=160, or x=1 and y=145, or x=95 and y=145, and (2) a fragmentof an amino acid sequence of (1), said fragment comprising at least 7contiguous amino acids from amino acid 96 through amino acid 196 of SEQID NO:2, said polypeptide or peptide containing a reactive epitope whichproduces at least one of a hundred and a cell-mediated response in a T.gondii infected host organism.
 9. A purified and isolated polypeptide orpeptide according to claim 2 containing a sequence selected from thegroup consisting of: (1) the amino acid sequence extending from an aminoacid position 95 to an amino acid position 145 of SEQ ID NO:2, (2) afragment of the amino acid sequence of (1) comprising at least 7contiguous amino acids from amino acid 96 through amino acid 145 of SEQID NO:2, (3) at least one of the amino acid sequences represented byGluProAspGluGlnGluGluValHisPheArgLysArgGlyValArgSerAspAlaGlu (SEQ ID NO3), or ArgLysArgGlyValArgSerAspAlaGluValThrAspAspAsnIleTyrGluGluHis (SEQID NO 4), orValThrAspAspAsnIleTyrGluGluHisThrAspArgLysValValProArgLysSer (SEQ ID NO5), or ThrAspArgLysValValProArgLysSerGluGlyLysArgSerPheLysAspLeuLeu (SEQID NO 6), or GluGlyLysArgSerPheLysAspLeuLeuLysLysLeuAlaLeuPro (SEQ ID NO7), and said polypeptide or peptide containing a reactive epitope whichproduces at least one of a humoral and a cell-mediated immune responsein a T. gondii infected host organism.
 10. A purified and isolatedpolypeptide or peptide according to claim 2 containing a sequenceselected from the group consisting of: (1) the amino acid sequenceextending from an amino acid position 95 to an amino acid position 145of SEQ ID NO:2, and (2) a fragment of the amino acid sequence of (1)comprising at least 7 contiguous amino acids from amino acid 96 throughamino acid 145 of SEQ ID NO:2, said polypeptide or peptide containing areactive epitope which produces at least one of a humoral and acell-mediated immune response in a T. gondii infected host organism. 11.A fusion protein comprising a polypeptide or peptide according to claim1, linked to a heterologous polypeptide sequence.
 12. A fusion proteincomprising a polypeptide or peptide according to claim 2, linked to aheterologous polypeptide sequence.
 13. A fusion protein comprising apolypeptide or peptide according to claim 8, linked to a heterologouspolypeptide sequence.
 14. A fusion protein comprising a polypeptide orpeptide according to claim 9, linked to a heterologous polypeptidesequence.
 15. A fusion protein comprising a polypeptide or peptideaccording to claim 10, linked to a heterologous polypeptide sequence.16. A method of detecting antibodies to T. gondii in a sample comprisingcontacting said sample with at least one polypeptide or peptideaccording to claim 2 under conditions sufficient to form animmunological complex between said polypeptide or peptide and saidantibodies, if present, and detecting said immunological complex.
 17. Amethod of detecting antibodies to T. gondii in a sample comprisingcontacting said sample with at least one polypeptide or peptideaccording to claim 8 under conditions sufficient to form animmunological complex between said polypeptide or peptide and saidantibodies, if present, and detecting said immunological complex.
 18. Amethod of detecting antibodies to T. gondii in a sample comprisingcontacting said sample with at least one polypeptide or peptideaccording to claim 9 under conditions sufficient to form animmunological complex between said polypeptide or peptide and saidantibodies, if present, and detecting said immunological complex.
 19. Amethod of detecting antibodies to T. gondii in a sample comprisingcontacting said sample with at least one polypeptide or peptideaccording to claim 10 under conditions sufficient to form animmunological complex between said polypeptide or peptide and saidantibodies, if present, and detecting said immunological complex. 20.The method of claim 16 wherein said sample is a biological sample froman individual and said method further comprises contacting said samplewith at least one further T. gondii polypeptide or peptide for measuringthe cellular immune response of said individual.
 21. The method of claim17 wherein said sample is a biological sample from an individual andsaid method further comprises contacting said sample with at least onefurther T. gondii polypeptide or peptide for measuring the cellularimmune response of said individual.
 22. The method of claim 18 whereinsaid sample is a biological sample from an individual and said methodfurther comprises contacting said sample with at least one further T.gondii polypeptide or peptide for measuring the cellular immune responseof said individual.
 23. The method of claim 19 wherein said sample is abiological sample from an individual and said method further comprisescontacting said sample with at least one further T. gondii polypeptideor peptide for measuring the cellular immune response of saidindividual.
 24. A kit for detecting anti-T. gondii antibodies in asample, said kit comprising: at least one polypeptide or peptideaccording to claim 2, optionally in combination with other polypeptidesor peptides being also optionally immobilized of a solid support,optionally, a buffer, or components necessary for producing the buffer,enabling a binding reaction to occur between the antibodies present insaid sample and said polypeptide or peptide, optionally, a means fordetecting the immune complex formed, and, optionally, an automatedscanning and interpretation device for inferring the presence of saidantibodies in said sample.
 25. A kit for detecting anti-T. gondiiantibodies in a sample, said kit comprising: at least one polypeptide orpeptide according to claim 8, optionally in combination with otherpolypeptides or peptides being also optionally immobilized of a solidsupport, optionally, a buffer, or components necessary for producing thebuffer, enabling a binding reaction to occur between the antibodiespresent in said sample and said polypeptide or peptide, optionally, ameans for detecting the immune complex formed, and, optionally, anautomated scanning and interpretation device for inferring the presenceof said antibodies in said sample.
 26. A kit for detecting anti-T.gondii antibodies in a sample, said kit comprising: at least onepolypeptide or peptide according to claim 9, optionally in combinationwith other polypeptides or peptides being also optionally immobilized ofa solid support, optionally, a buffer, or components necessary forproducing the buffer, enabling a binding reaction to occur between theantibodies present in said sample and said polypeptide or peptide,optionally, a means for detecting the immune complex formed, and,optionally, an automated scanning and interpretation device forinferring the presence of said antibodies in said sample.
 27. A kit fordetecting anti-T. gondii antibodies in a sample, said kit comprising: atleast one polypeptide or peptide according to claim 10, optionally incombination with other polypeptides or peptides being also optionallyimmobilized of a solid support, optionally, a buffer, or componentsnecessary for producing the buffer, enabling a binding reaction to occurbetween the antibodies present in said sample and said polypeptide orpeptide, optionally, a means for detecting the immune complex formed,and, optionally, an automated scanning and interpretation device forinferring the presence of said antibodies in said sample.
 28. A kitaccording to claim 24, further comprising at least one non-T. gondiipathogen-specific detection reactant selected from the group consistingof a non-T. gondii, pathogen-specific, polypeptide, antibody andpolynucleic acid.
 29. A kit according to claim 25, further comprising atleast one non-T. gondii pathogen-specific detection reactant selectedfrom the group consisting of a non-T. gondii, pathogen-specific,polypeptide, antibody and polynucleic acid.
 30. A kit according to claim26, further comprising at least one non-T. gondii pathogen-specificdetection reactant selected from the group consisting of a non-T.gondii, pathogen-specific, polypeptide, antibody and polynucleic acid.31. A kit according to claim 27, further comprising at least one non-T.gondii pathogen-specific detection reactant selected from the groupconsisting of a non-T. gondii, pathogen-specific, polypeptide, antibodyand polynucleic acid.